Microscopy,Immunohistochemistry, andAntigen Retrieval Methods

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Microscopy,Immunohistochemistry, andAntigen Retrieval Methods

For Light and Electron Microscopy

M. A. HayatKean University

Union, New Jersey

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Preface

There are several important reasons for publishing this book. One reason is to presentchemical and physical principles governing the processing of tissues using microwaveheating as an adjunct to fixation, embedding in a resin, and staining. A second reason is topoint interested readers to a number of recent developments in the retrieval and localiza-tion of antigens in normal and pathological tissues. The greatest concentration of work inthis field has focused on the detectability of disease-related proteins. Therefore, as exam-ples, the detectability and the role in disease of estrogens, p53, p185, Ki-67, and PCNA arediscussed in detail.

A third reason is to review favorable aspects of the histochemical approach, wherebyit yields data not obtainable by any other means, including biochemical assays.Histochemistry, for instance, contributes to acquiring knowledge about the biological activ-ity of normal and diseased cells, which is supported by illustrations. Immunohistochemistrydefines the function of cell types in a tissue and organs by localizing and identifying theircontents or products. This methodology is highly visual; illustrations, especially colorimages, often contribute as much to correct understanding and interpretation of the resultsas the text. Therefore, the results of many methods are illustrated.

During the last decade there has been significant progress in understanding themechanisms responsible for antigen masking during fixation and subsequent unmasking,primarily by heating or, in some cases, by enzymatic digestion. Comparative studiesdemonstrate, for example, that not only microwave heating but also other sources ofheating are effective in antigen retrieval. Similar studies also indicate that althoughsodium citrate buffer is in common use as the antigen retrieval fluid, unmasking of certainantigens requires other fluids. These and other new developments are discussed in thisvolume.

In preparing the reader to study the location of proteins and carbohydrates, it is nec-essary to explain the advantages and limitations of the study. A potential limitation of theimmunohistochemical approach arises from the possibility of false-negative staining dueto the failure of an antibody to yield positive results. It is equally important to be aware ofthe possibility of false-positive staining, which can arise if the method is not scrupulousregarding histochemical negative and positive controls. Suggestions are offered to at leastminimize these histological artifacts. In this regard the importance of negative and positivecontrols cannot be overemphasized. Negative controls involve the omission of the primary

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viii Preface

antibody with an immunoglobulin that is directed against an unrelated antigen. Thisimmunoglobulin must be of the same class, source, and species. Positive controls involvethe use of a tissue section of known positivity. The absence of staining in a test tissuesection does not necessarily indicate that the antigen is not present. It should be noted thatsome antigens are present not only in pathological tissues but also in healthy tissues.

The arrival of methods and instruments to investigate disease processes at the molec-ular level induces pathologists to apply these new procedures to existing problems of dis-ease pathogenesis and disease evolution and offers clues to therapeutic intervention. Suchmethods include histological microdissection (in conjunction with real-time quantitativereverse transcriptase–polymerase chain reaction), cDNA microarray, anticancer vaccines,and gene regulation (genes can be turned on and off). Some of these techniques are sum-marized in Chapter 1. The limited space available did not allow a detailed presentation ofthe expanding world of molecular pathology.

Chapter 1 contains seemingly diverse topics, but all are related to immunopathology.It is my hope that pathologists will benefit from these step-by-step protocols, which arepresented in a self-explanatory form so that the reader can practice them without outsidehelp. Chapter 8 contains details of specific methods because the various parameters of pro-cessing of each type of antibody, antigen, and tissue may need to be varied to obtain opti-mal results. I have tried to synthesize a large number and variety of immunohistochemicaltechniques into a single and concise basic handbook. Some alternative methods are alsoincluded.

I have explained not only how to use a technique but also why to use it—along withits advantages and limitations. An example is the microwave heating methodology. Themethods presented were carefully selected and are reproducible but can be modified,depending on the objective of the study. Moreover, as the antigen retrieval methodology isrelatively new, it requires fine-tuning.

There is a degree of necessary repetition in some chapters, which allows them tostand alone. This approach helps the reader to carry out a procedure where it is describedwithout searching for its details somewhere else in the book. Cross-reference of informa-tion among chapters, wherever possible, is given.

Where possible, commercial sources of reagents, kits, and equipment are listedthroughout the text instead of in a separate index. Extensive references are provided tofacilitate the task for those readers who may wish to consult the literature for additionalinformation on specific topics. All books can be improved, and this volume is no excep-tion. I welcome constructive criticism from my colleagues and students. With this help Ilook forward to offering a greatly improved second edition.

The writing of this book would not have been possible without the most generoushelp of a large number of distinguished scientists. I am very grateful for the thoughtful andinvaluable suggestions and illustrations received from scientists throughout the world. Itis appropriate to acknowledge significant contributions made to the understanding andpractice of antigen retrieval methodology and applications of microwave heating byHector Battifora, Mathilde E. Boon, Giorgio Cattoretti, Richard J. Cote, Ann M. Dvorak,Jules M. Elias, Johannes Gerdes, David Hopwood, Allen M. Gown, Richard Horobin,L. P. Kok, Anthony S.-Y. Leong, Gary R. Login, Enrico Marani, Shan-Rong Shi, Albert J. H.Suurmeijer, and Clive R. Taylor. It is not possible to mention all the scientists who haveplayed a role in the development of this technology.

Preface ix

The help and encouragement received from Dean Betty Barber throughout the writ-ing of this book are greatly appreciated and will be remembered. I thank Patricia Lemusand Elizabeth McGovern for their expert secretarial assistance in the preparation of themanuscript, and I appreciate the help and cooperation extended to me by Roberta Klarreich,the production editor, throughout the production of this volume.

M. A. HayatOctober 2001

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Contents

Chapter 1. Introduction 1

Cytogenetic Evaluation of TumorsGenetic Instability of TumorsTumor Heterogeneity

Histological Microdissection

Antitumor VaccinesMolecular GeneticscDNA Microarray TechnologyAngiogenesis

Vascular Endothelial Growth Factor

Immunohistochemical Localization of Vascular Endothelial

Growth Factor

Telepathology (Telemedicine)

Future of ImmunohistopathologyPreparation of Buffers

111214141517182023

24252829

Chapter 2. Antigens and Antibodies 31

AntigensEpitopes

AntibodiesPolyclonal Antibodies

Production of Polyclonal Antiserum

Affinity Chromatography

Monoclonal Antibodies

Specificity of Monoclonal Antibodies

MIB-1 Monoclonal Antibody

Production of Monoclonal Antibodies

Bivalent and Bispecific Monoclonal Antibodies in Cancer Therapy

Recombinant AntibodiesAnticancer Monoclonal Antibodies

31323334353537383941444647

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xii Contents

Antibody Cross-ReactivityPolyreactive AntibodiesCommercial Sources of Antibodies

484950

Chapter 3. Fixation and Embedding 53

FormaldehydeNature of Formaldehyde Solution

Mechanism of Fixation with Formaldehyde

Comparison of Formaldehyde with Glutaraldehyde

Fixation with Formaldehyde

Effect of Prolonged Fixation with Formaldehyde

Formalin Substitute Fixatives

Fixation Conditions

Effect of Heating on Fixation with GlutaraldehydeMicrowave Heat–Assisted Fixation with Osmium TetroxideRole of Microwave Heating in Enzyme Cytochemistry

Fixation for Enzyme Cytochemistry Using Microwave at Relatively

Low Temperature

Cryopreservation in the Presence of Microwave HeatingParaffin Embedding

Paraffin Embedding in Microwave Oven

Paraffin Embedding in Vacuum-Microwave Oven

Microtomy of Paraffin-Embedded Tissues

Silanting of Glass Slides

Vacuum-Assisted Microwave Heating

5354545657585960616264

6465656767676869

Chapter 4. Factors Affecting Antigen Retrieval 71

FixationDenaturationHeatingpHMolarityAntigen Retrieval Fluids

Glycerin as Antigen Retrieval Fluid

Procedure

pH of Antigen Retrieval Fluids

Ionic Strength of Antigen Retrieval Fluids

Antibody PenetrationAntibody Dilution

Diluent Buffer for Primary Antibodies

Storage of Paraffin-Embedded TissuesStorage of Tissue SlidesSignal Amplification

71727374747577787879798082838489

Contents xiii

Tyramine Amplification Method

Preparation of Biotinylated Tyramine

Rolling Circle Amplification

909292

Chapter 5. Problems in Antigen Retrieval 95

Lack of ImmunostainingBackground StainingProblem of Endogenous Biotin

959698

Procedure

Mirror Image Complementary AntibodiesProcedure

Fixation of Frozen TissuesHot Spots (Areas) in Microwave OvenProblem of Antigen Retrieval StandardizationTest BatteryIntraobserver and Interobserver Variation in DiagnosisQuantitation of ImmunostainingAutostainers

Capillary Gap Stainers

Centrifugal Stainers

Flat-Method Stainers

Volume-Corrected Mitotic IndexThe Gleason Grading SystemUniversal Antigen Retrieval Method?Calibration of Microwave Oven

100101101102102103104105105107109109109110111113114

Chapter 6. Antigen Retrieval 117

Possible Mechanisms of Antigen RetrievalNonthermal Effects of Microwave Heating

Effect of Endogenous Calcium on Antigen MaskingUse of Ethylenediaminetetraacetic Acid (EDTA)

for Antigen RetrievalAntigen Retrieval with Heat TreatmentAdvantages of HeatingHeating Methods

Mechanism of Epitope Retrieval by Microwave Heating

Duration of Microwave Heating

Antigen Retrieval in a High-Pressure Microwave Oven

Antigen Retrieval at Low Temperature

Use of Heat for Staining

Rapid Immunostaining of Frozen Sections

Enhanced Polymer One-Step Staining Procedure

Modified En Vision Procedure

Hazards and Precautions in the Use of Microwave Ovens

117119120

123124124125130131132132136138139139141

xiv Contents

Limitations of Microwave Heating

Wet Autoclave MethodProcedure 1

Procedure 2

Ultrasound TreatmentProcedure

Nonheating MethodsDetergents

Procedures

Proteolytic Enzyme Digestion

Procedure

Enzyme Digestion and Relatively Low Temperature

(80°C)–Assisted Antigen Retrieval

Comparison of Antigen Retrieval Methods: A Summary

142145145146146148148148149151152

152153

Chapter 7. Antigen Retrieval on Resin Sections 155

Role of Fixative and Embedding Resin in Antigen RetrievalImmunostaining of Thin Resin SectionsAntigen Retrieval on Sections of Modified Epoxy ResinEffect of HeatingAntigen Retrieval on Thin Resin Sections Using AutoclavingRapid Staining of Thin Resin Sections in Microwave OvenMicrowave Heat–Assisted Rapid Processing of Tissues for Electron

MicroscopyMicrowave Heat–Assisted Immunolabeling of Resin-Embedded

SectionsMicrowave Heat–Assisted Immunogold Methods

Immunogold-Silver Staining

Droplet Procedure

156158160161161163

163

163167167167

Chapter 8. General Methods of Antigen Retrieval 169

General Procedure for Antigen Retrieval Using MicrowaveHeating

Antigen Retrieval in Archival TissuesMethod for Microwave Heating of Archival Tissue Blocks

Antigen Retrieval Using a Conventional OvenHot Plate–Assisted Antigen Retrieval

Procedure

Hot Plate–Assisted Grading of Vulvar Intraepithelial NeoplasiaWater Bath Heat–Assisted Antigen Retrieval

Procedure for Electron Microscopy

Procedure for Light Microscopy

Procedure for Free-Floating Sections

Microwave Heat–Assisted Evaluation of Global DNAHypomethylation

169173174175175175176177178178180

180

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Contents xv

Procedure

Microwave Heat–Assisted Enhanced PeroxidaseOne-Step Method

Procedure

Microwave Heat–Assisted Immunostaining of Cell SmearsDouble Immunostaining Using Microwave HeatingMicrowave Heat–Enhanced Double Immunostaining of Nuclear

and Cytoplasmic AntigensProcedure

Microwave Heat–Assisted Immunohistochemical Localization ofCyclin D1

Microwave Heat–Assisted Immunofluorescence Staining of TissueSections

Procedure

Microwave Heat–Assisted Double Immunofluorescence LabelingProcedure

Microwave Heat–Assisted Double Indirect ImmunofluorescenceStaining

Procedure

Control Procedures

Immunoenzymatic Detection

Combined Microwave Heating and Ultrasound AntigenRetrieval Method

Combined Enzyme Digestion and Microwave HeatingAntigen Retrieval Method

Pressure Cooker–EDTA–Assisted Antigen Retrieval2-Mercaptoethanol–Sodium Iodoacetate–Assisted

Antigen RetrievalAntigen Retrieval with Steam–EDTA–Protease Method

Procedure

Picric Acid–Steam Autoclaving–Formic Acid–GuanidineThiocyanate–Assisted Retrieval of Prion Protein

Procedure

Simultaneous Detection of Multiple AntigensProcedure

Use of Multiple Antibodies for Labeling AntigensProcedure

Antigen Retrieval in Neuronal Tissue Slices before VibratomeSectioning

Microwave Heat–Assisted Antigen Retrieval in Freshly FrozenBrain Tissue

Procedure

Microwave Heat–Assisted Rapid Immunostaining of FrozenSections

Procedure

Microwave Heat–Assisted Immunocytochemistry of ThinCryosections

Procedure

181

181181182182

183183

184

185186186186

187187188189

189

190191

191192192

192194194196196197

198

198199

199200

200201

xvi Contents

Pressure Cooker–Assisted Detection of Apoptotic CellsImmunohistochemical Localization of Prostate-Specific AntigenImmunohistochemistry

Procedure

201202203203

Chapter 9. Other Applications of Microwave Heating 205

Carbohydrate AntigensOvarian Carcinoma

Microwave Heat–Assisted Carbohydrate Antigen Retrieval

Enzyme Digestion–Assisted Carbohydrate Antigen Retrieval

Nucleolar Organizer–Associated Region ProteinsProcedure

Nucleolar Size

In Situ HybridizationRadioactive Probes

Nonradioactive Probes

Enhancement of in Situ Hybridization Signal with Microwave Heating

Procedure for in Situ Hybridization of DNA

In Situ Hybridization of RNA in Skeletal Tissues

Microwave Heating for in Situ Hybridization of mRNA in

Plant Tissues

Microwave Treatment

StainingMicrowave Heat–Assisted Fluorescence in Situ Hybridization

Procedure for Gastrointestinal Neoplasia

Nuclear Fluorescence in Situ Hybridization Signal Using

Microwave Heating

Microwave Heat–Assisted Polymerase Chain ReactionProcedure

Detection of Antigens by Flow CytometryMicrowave Heat–Assisted Flow Cytometry

Procedure 1

Procedure 2

Microwave Heat–Assisted Enzyme-Linked Immunosorbent AssayMicrowave Heat–Assisted Scanning Electron MicroscopyMicrowave Heat–Assisted Confocal Scanning MicroscopyMicrowave Heat–Assisted Correlative Microscopy

Procedure

205206208208209211212213215215217218219

221221221222222

223224224225225227228228229230230230

Chapter 10. Cell Proliferating Antigens 233

Ki-67 AntigenImmunohistochemistry

Limitations of Immunohistochemistry

Antibodies

233235237237

Contents

Recent Applications of MIB-1 Antibody

Ki-67 Antigen Retrieval Using Microwave Heating

Ki-67 Antigen Retrieval Using Autoclave Treatment

Proliferating Cell Nuclear AntigenImmunohistochemistry

Limitations of PCNA Immunohistochemistry

Immunostaining of PCNA on Cryostat Sections

p53 AntigenWild-Type p53 Protein

Mutant p53 Protein

p73Antibodies

Examples of Antibody Dilutions

Immunohistochemistry

Use of Multiple Antibodies for Labeling p53 AntigenWild-Type p53 Antigen Retrieval Using Microwave Heating

p53 Antigen Retrieval Using Microwave Heating

Frozen Section Immunohistochemistry of p53

239240240241242243245245247248249250253253256257257258

xvii

Chapter 11. Estrogens 261

Estrogen ReceptorsEstrogen Receptor Alpha

Estrogen Receptor Beta

Estrogen Receptor Gamma

Distribution of Estrogen Receptors

Role of Estrogen Receptors in Breast CancerBreast Cancer and Tamoxifen

AntibodiesImmunohistochemistryComparison of Immunohistochemistry with Biochemical

Ligand-Binding AssaysDextran-Coated Charcoal Assay

Semiquantitative Assessment of Estrogen ReceptorsImmunostaining of Estrogen Receptors in Prostate TissueImmunostaining of Estrogen and Progesterone Receptors in

Fine-Needle Aspirates of Breast

262265267268268269270270273

275276276277

278

Chapter 12. HER-2 (c-erbB-2) Oncoprotein 281

HER-2/neu OncogeneHER-2 OncoproteinHER-2 OverexpressionSimultaneous Overexpression of HER-2 and p53Distribution of HER-2 in Carcinomas

281283284284285

xviii Contents

Astrocytic Tumors

Bladder Carcinoma

Ewing’s Sarcoma

Intrahepatic Cholangiocellular Carcinoma

Laryngeal Squamous Cell Carcinoma

Non-Small-Cell Lung Carcinoma

Ovarian Carcinoma

Prostate Carcinoma

Squamous Cell Carcinoma of Cervix

Methods for Detecting HER-2 StatusQuantitative Analysis of HER-2/neu Gene ExpressionDetection of HER-2 OncoproteinBispecific Antibodies

Bispecific Antibody MDX-H210

VaccinesGenetic Immunization

ImmunohistochemistryHerceptin (Trastuzumab)HercepTest

Controls and Scoring System

Immunostaining of HER-2 Protein Using HercepTest

285285285286286286287288288289290291293294295295296297299300302

References 305

Index 351

Microscopy,Immunohistochemistry, andAntigen Retrieval Methods

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Chapter 1

Introduction

Immunohistochemistry and immunocytochemistry have played an important role in thefields of cell and tissue biology, embryology, and diagnostic pathology. These methodolo-gies facilitate precise analysis of the chemistry of cells and tissues in relation to structuralorganization. The information derived from these techniques will continue to contributeto our understanding of dynamic molecular, cellular, and pathological processes. Forexample, immunohistochemistry has revolutionized the field of tumor diagnosis and hasprovided a powerful tool for pathologists to better characterize difficult or unusualneoplasms. In addition, this technology has provided information that has resulted in thereclassification of many neoplasms and, in some cases, the creation of new categories thatwere previously unrecognized. In this volume the emphasis is on the application of thismethodology to routine diagnostic pathology.

The immunohistochemical method localizes and identifies a specific antigen in a cellor a tissue specimen. In the most common approach, specimens are fixed in 10% neutralbuffered formalin and embedded in paraffin. Sections ( thick) are deparaffinizedwith xylene and then rehydrated in a series of ethanol solutions with decreasing concen-trations. After drying, sections are treated with 3% hydrogen peroxide to block endoge-nous peroxidase. If required, sections are subjected to antigen retrieval. Nonspecificimmunostaining is blocked by treating the sections with 10% normal serum. The primaryantibody applied is either monoclonal or polyclonal. A secondary antibody, linked with animaging system (usually a peroxidase), is applied to recognize the primary antibody.

Contrary to the popular use of the term antigen retrieval in the literature and by com-mercial companies, the correct term is epitope retrieval, since it is the epitope (antigenicdeterminant) that is recognized by the antibody (paratope) instead of antigen molecule asa whole. A monoclonal antibody reacts with a specific region of the antigen irrespective ofthe conformation of other regions of the same antigen. Different monoclonal antibodiesgenerally react with different epitopes of the same antigen. Thus, the epitope is the part ofthe antigen to which the antibody is directed. An epitope indeed defines antigen specificity.Furthermore, the fact that monoclonal antibodies to different epitopes of the same antigenmolecule behave differently in “antigen retrieval” indicates that what is being retrieved isan epitope. In other words, two different epitopes of the same antigen may require differ-ent treatments for their accessibility to the respective antibodies. This is the context inwhich this subject should be understood.

1

2 Chapter 1

The other point of view, which favors the term antigen retrieval instead of epitoperetrieval, is as follows (S.-R. Shi, personal communication). It is likely that the mechanismof antigen retrieval is based on chemical modification of protein conformation. Therefore,retrieval of formalin-modified (or masked) antigenicity must be a restoration of the proteinstructure, as any antigen/antibody recognition is dependent on protein conformation. Thisis particularly true for discontinuous epitopes (most antigen determinants are discontinu-ous epitopes), which consist of amino acid sequences apart from each other on onepolypeptide (or actually located on distinct polypeptides) but brought near each other inthe tertiary or quaternary structure of the protein. In other words, restoration of the func-tion of an epitope (antigenicity) is the retrieval of its protein conformation, i.e., retrieval ofantigen. Because the concept of epitope is not an intrinsic feature of a protein existingindependently of its paratope partner, the term epitope refers to only a functional unit, butnot a stoic structure of the protein. Shi et al. (2000a) have further justified the use of theterm antigen and rejected the relevancy of the term epitope in immunohistochemistry.

In support of Dr. Shi’s opinion is the fact that in some cases the absolute specificityof even monoclonal antibodies can be questioned. The absorption control cannot alwaysdetermine whether the protein bound in the tissue is the same protein used for absorption.The monoclonal antibody may instead recognize a similar epitope of an unrelated protein,especially following tissue fixation. Absorption controls therefore may not provide thespecificity of the antibody for a protein under study in the tissue.

In light of the above-mentioned difference of opinion, and in the absence of a definiteunderstanding regarding unmasking of an epitope or whole antigen molecule as a result ofunmasking treatments (heat or nonheat treatment), both terms, epitopes and antigens, areused in this volume.

Immunohistochemistry has surpassed other techniques in its effectiveness in thein situ preservation and detection of antigens. Immunopathology has become a valuable oreven an essential adjunct to diagnostic pathology. It is affirmed that diagnostic immuno-histochemistry is indispensable in surgical pathology for diagnosis, therapy, and progno-sis. The usefulness of this methodology depends and will continue to rely on three majorfactors: (1) availability of specific primary antibodies; (2) an efficient detection system;and (3) correct interpretation and significance of the findings. An increasing number ofmonoclonal antibodies is being produced, and many of them are commercially available;these sources are given in Chapter 2. The role of antibodies in diagnostic pathology is dis-cussed in this chapter. Highly sensitive detection systems are available, and their signalsare being continuously enhanced to achieve high signal-to-noise ratio. Methods of scoringthe signals are also available. These improvements are discussed in Chapter 4.

All immunohistological methods depend on the successful completion of a series ofsequential steps, beginning with specimen collection; their morphological and antigenicpreservation (chemical fixation or rapid freezing) or antigenic retrieval; incubation in anantibody or sequence of antibodies; staining; signal counting; and interpretation of results.Each of these steps must be performed as efficiently and correctly as possible. If even oneof these steps is suboptimal, the remaining steps, even though perfectly carried out, cannever compensate for the inefficient step. Any error in immunohistochemistry, especiallywhen applied to clinical diagnosis, is unacceptable. This methodology is not only a sciencebut also an art; each aspect depends on the other.

Progress in molecular biology is intimately related to advancement in technology.One fundamental goal of cell research is to understand the functions of molecules that

Introduction 3

constitute cells and tissues. This understanding can be enhanced by examining the molec-ular details and subcellular location of cell components. The precise extracellular andintracellular localization of molecules under different physiological and pathological con-ditions yields clues to their possible functions. These aspects of antigen molecules andreceptors, especially clinically important ones such as p53, Ki-67, PCNA, p185, and estro-gens, are discussed in detail in Chapters 10, 11, and 12.

The achievement of the above-mentioned goal received an impetus from the devel-opment of the heating methodology (especially microwave heating) for antigen retrieval.Microwave heating was introduced into biomedical research approximately two decadesago for the rapid processing of plant and animal tissues. The development of this techniquewas a significant step forward in the application of histochemistry, immunohistochemistry,and immunocytochemistry. In other words, this methodology has significantly contributedto the localization of macromolecules and molecules (including antigens) and thus to anunderstanding of their functions. This technique is also useful for enhancing the detectionof RNA and DNA by in situ hybridization (see page 213). Another example of the appli-cation of microwave heating is in conjunction with flow cytometry (see page 225). Thepolymerase chain reaction (PCR) has also been used in conjunction with microwaveheating for studying DNA (see page 224). Yet another application of microwave heating iswith the enzyme-linked-immunosorbent assay method (ELISA) (see page 228). Tissuecryopreservation with diminished ice crystal growth has also been accomplished with thisversatile technology (see page 65). Application of microwave heating to enzyme cyto-chemistry, autoradiography, and X-ray microanalysis has been attempted (Mizuhira andHasegawa, 1996).

Significant aspects of the basic biology of disease processes are now assuming clini-cal importance in diagnosis and prognosis. The pathologist can identify an ever-increasingrange of antigens in tissue sections using the techniques mentioned above. Identificationof tissue antigens using these methods is of fundamental importance for clarifying tumorproteins or carbohydrates, determining the diagnosis and prognosis of tumors, character-izing pervasive nepotistic alterations in tissues such as prostate, subclassifying neoplasms,evaluating the response of tumors and pervasive nepotistic changes to certain therapies(i.e., as a surrogate intermediate and end point), selecting patients who are candidates forspecific therapies (e.g., immunotherapy), and identifying pathogenic organisms (Arnoldet al., 1996). For these and other reasons, immunohistochemistry has become the mostimportant tool in research and diagnostic pathology. It permits detection of defined anti-gens on cryostat, paraffin, and resin sections of normal and diseased tissues.

Immunohistochemical and immunocytochemical localization of antigens is a power-ful tool that provides insight into some of the salient features of cell and tissue complex-ity. Such studies, for example, demonstrate relationships between normal cell structure andfunction and pathological consequences. Presently, immunohistochemistry is firmly estab-lished as the most important method for detecting antigens with the light microscope. Itcan be effectively used to examine various antigens in the sections of formaldehyde-fixedand paraffin-embedded tissues (Hayat, 2000a). The availability of the equipment to carryout immunohistochemistry and the introduction of many new monoclonal antibodies makeit possible to apply this technique to retrospective studies.

The introduction of a large number of new monoclonal antibodies of improved sensi-tivity and specificity, which are available in ready-to-use kits, has made possible a wideruse of immunohistochemistry for antigen analysis. In addition, the development of various

4 Chapter 1

antigen retrieval methods during the past two decades has enabled many more antibodiesto access antigens that were undetectable or minimally detectable in the past. Today almostany antigen that survives tissue processing has the potential to be localized immunohisto-chemically. As a result of these methods, additional antibodies have become paraffin- andresin-compatible, which permits heat-treated tissue sections to be used for detecting anti-gens with the light and electron microscopes.

New antigen retrieval methods, especially microwave heating and other heating pro-cedures and ultrasound treatment, can effectively retrieve antigens from tissues left informaldehyde for prolonged periods. The introduction of the computer-assisted image ana-lyzer and other automated equipment (e.g., the automatic stainer), and generation of anti-bodies to synthetic peptides, have ushered immunohistochemistry into a higher level ofefficiency, accuracy, and quantitation. The demand for a more precise spatial localizationof epitopes favors the use of antibody fragments (e.g., Fab), peptides, or ligands.

These advances facilitate the use of antigen detection for correct diagnosis and prog-nosis. Furthermore, advances in detection accuracy provide guidelines to study andunderstand more complicated biological problems. To achieve these goals, standardizationof antigen retrieval methods is necessary, at the least, to minimize inter- and intralabora-tory (including interobserver) variability of immunostaining (see Chapter 5). However,even in the absence of such standardization, the method has become the most effective toolin light microscope immunohistochemistry and, to some extent, in electron microscopeimmunocytochemistry.

Introduction 5

Although antigen retrieval is carried out most commonly on paraffin sections, it canalso be accomplished on semithin or thin resin sections for light and electron microscopy,respectively. Thin sections of routinely used resins such as epoxy, LR White, LR Gold,and Lowicryls can be used for detecting antigens with the light or electron microscope.These resins, in conjunction with microwave heating, can also be used for cell and ultra-structural studies with the light microscope, and scanning and transmission electronmicroscope (Fig. 1.1).

This procedure can also be employed for studying bacteria with the scanning elec-tron microscope (Fig. 1.2).

The advantages of resin sections include better preservation of cellular details, assist-ing the achievement of higher resolution, and the ability to carry out correlative studies ofthe same tissue with the light microscope and scanning and transmission electron micro-scope (Fig. 1.3). In addition, resin sections (also cryosections) allow immunogold andsilver-enhanced immunogold staining.

Biochemical assays such as the dextran-coated charcoal (DCC) assay, certain signalamplification techniques, and other cytosol-based methods have been mostly replaced

6 Chapter 1

by immunohistochemistry because the former methods are costly and often difficult toreproduce. For example, the DCC assay requires rather large tissue specimens and may beadversely influenced by tissue heterogeneity (e.g., tumors), presence of bound endogenousestrogen, and sampling error (Hendricks and Wilkinson, 1993). In cytosol-based biochem-ical assays, tissues are indiscriminately homogenized (e.g., tumor, stroma, inflammatorycells, and epithelial cells). Therefore, the results expressed in fentomoles per milligram ofthe total protein are variably diluted because of the presence of nontumorous cells.

In contrast, immunohistochemistry can be carried out with smaller tissue specimensand is less affected by tissue heterogeneity or endogenous hormones. This techniqueallows direct histological visualization, which permits separation of tumors from stroma,inflammatory cells, and normal cells. However, the DCC assay can be employed for cor-relative studies; therefore, the selection of a particular method in a diagnostic laboratoryshould depend on a number of factors, including specificity, sensitivity, rapidity, use ofpotentially harmful reagents, availability of equipment, cost, and application to a widerange of antigens.

However, the central problem in immunohistochemistry is to retain antigenicity with-out sacrificing the quality of cell morphological preservation. It has been established thatthe preservation of antigenicity is inversely related to the preservation of cell morphology(Hayat, 2000a); thus, tissue preparation methods optimal for the preservation of cell mor-phology introduce protein crosslinking and are therefore suboptimal for preserving anti-genicity. Because preserving antigenicity in immunohistochemistry is more important thanpreserving morphology, 10% formaldehyde is generally used for fixation. Glutaraldehyde,on the other hand, yields excellent ultrastructural preservation but severely masks mostantigens by introducing irreversible protein crosslinking. The mechanism(s) responsible

Introduction 7

8 Chapter 1

for the antigen-masking effects of fixatives are discussed in Chapter 4. Tissue embeddingin paraffin also adversely affects antigen detection.

The alternative to chemical fixation and paraffin embedding is to use frozen sectionsof fresh-frozen tissues (snap freezing). These cryostat sections tend to provide improvedantigen detection. Proteins are retained in these sections at least until the cryosections areplaced into aqueous incubation and staining solutions. Most antibodies recognize antigensbetter in frozen sections than in sections of chemically fixed and paraffin-embedded tissues.Fixation of frozen sections has also been recommended by some workers to immobilizeproteins during subsequent processing of cryostat sections and thus improve the antigenlocalization. However, an agreement on the beneficial effect of this practice is lacking.Moreover, cryostat sections are difficult to prepare, and the quality of morphological preser-vation is comparatively poor (Fig. 1.4).

Fortunately, methods are available that satisfactorily preserve cell morphology aswell as antigenicity. The best compromise is to use a mixture of 6% formaldehyde and glu-taraldehyde of a low concentration (0.1–0.4%). This approach, which is used extensivelyfor electron microscopy, should be used for light microscopy and is presented in thisvolume. The optimal immunohistochemical method should ideally detect all specific epi-topes, but in practice this is not possible. The best that can be accomplished is to optimizethe protocol to detect all the detectable epitopes.

Presently, the majority of the antigen retrieval studies are carried out using microwaveheating, although other heating methods such as autoclaving (page 145) or pressure cooking(pages 127–128) can be equally effective, depending on the types of tissue and antigenunder study. Antigen retrieval can also be accomplished in a microwave oven under vac-uum, as can rapid fixation, resin embedding, and staining. Ultrafast (milliseconds) or fast(seconds to minutes) fixation can be achieved with this method. Even rapid fixation withosmium tetroxide can be carried out in a microwave oven (see Chapter 3).

Like any other technique, microwave heating has certain limitations. A well-knownexample is the uneven distribution of hot and cold spots in the oven, although this can beminimized by using a water load in the oven. Prior to placing the water load in the oven,cold and hot spots should be located in the oven cavity by using a high-brightness neonbulb array. Hot spots must be avoided because an excessive increase in temperature dur-ing irradiation is a major cause of poor fixation. The problem of hot spots apparently canbe avoided by using heating methods other than microwave heating. One should also beaware that microwave heating may promote cross-reactivity by producing or unmaskingantigens closely related to those under study. The result may be the decreased specificityof an established and generally available antibody (Alexander and Dayal, 1997). Otherlimitations of microwave heating are listed on pages 142-144.

Antigen retrieval can also be accomplished by treating paraffin sections with diges-tive enzymes. A number of proteolytic enzymes, including trypsin, pepsin, pronase, andficin (a plant enzyme), have been used for unmasking antigens. However, effectiveness ofeach enzyme is limited to a few types of antigens. Moreover, enzymatic digestion is inef-fective for antigen retrieved in overfixed tissues and tends to damage cell morphology,especially when the treatment is prolonged. Cumulative evidence indicates that heating isgenerally better than digestion. Therefore, the latter approach is not preferred unless theformer is unsuccessful. Better results are obtained in some cases when enzyme digestionis used in conjunction with heat treatment.

Intro

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10 Chapter 1

Immunohistological diagnosis is critically linked to an assessment of the morpholog-ical appearance of the cell. This is accomplished by employing a panel of monoclonal anti-bodies to establish the immunoprofile of a tumor and, it is hoped, to minimize the risk offalse-negative or false-positive staining. Therefore, a thorough knowledge of the chemistryof tissue processing, including antigen retrieval, is essential for routine practice. The anti-bodies described in this volume are immunoreactive in fixed, paraffin-embedded tissuesections and therefore are the mainstay of routine diagnostic histopathology. However, itshould be noted that the specificity of certain monoclonal antibodies, as commonly under-stood, is not valid because such an antibody shows cross-reactivity, that is, binding toepitopes shared by related proteins in the same tissue or from different tissues.

Finally, the use of antibodies to clinically important antigens for diagnostic and prog-nosis purposes requires a complete understanding of the role of antigens in the biology ofdisease. For correct interpretation of histological findings, the pathologist must be knowl-edgeable about pathophysiology, notwithstanding the availability of the most specificpanel of monoclonal antibodies. For this and other reasons the role of several antigens, inhealth and disease is discussed in detail in Chapters 10, 11, and 12. The following discus-sion summarizes the role of estrogens, p53, Ki-67, PCNA, and p185 proteins in health anddisease.

The importance of estrogens in health and disease becomes apparent when one con-siders that these hormones trigger and govern essential functions such as growth, differ-entiation, and the functioning of many target tissues. They also significantly influence theproliferative and metastatic states of breast cancer cells. It is also known that estrogensaffect the regulation of gene transcription through interaction with at least two estrogenreceptors ( and ). The role of estrogens and their receptors in breast cancer isemphasized in this volume. As examples, immunohistochemical localization of thesereceptors in the prostate tissue and in fine-needle aspirates of breast is presented.

The tumor suppressor gene p53 encodes a nuclear phosphoprotein which is expressedin most, if not all, tissues of the body. The steady-state levels of this protein in normalsomatic cells are usually very small because this newly synthesized protein is highly sen-sitive to ubiquitin/proteasome-mediated degradation, preventing its accumulation in cells.It functions primarily as a transcription factor that is activated in response to genotoxicstress, including DNA damage. Thus it controls the expression of many genes involved inregulating the cell cycle and apoptosis. In this way, p53 prevents the excessive accumula-tion of mutations and harmful cells which could give rise to malignancies. On the otherhand, mutation of p53 occurs frequently in human oncogenes. These mutations in tumorsabrogate the regulatory function of p53 on the cell cycle and lead to increased half-life ofthe otherwise very unstable wild-type p53 protein. The importance of immunohistochem-ical localization of p53 protein becomes obvious when considering that approximately50% of all human malignancies exhibit mutation and aberrant expression of this protein,making it an important target candidate for cancer immunotherapy. Details of immunohis-tochemical localization of wild-type p53 and mutant p53 are explained in this volume.

Ki-67 is a nuclear and nucleolar phosphoprotein. Although the biological function ofKi-67 has not been fully elucidated, it is accepted that this antigen, along with p53, PCNA,cyclin Dl, and bc12, plays an important role in regulating somatic cell proliferation.Immunohistochemical examination using a Ki-67 labeling index is a promising prolifera-tion marker, as a higher rate of Ki-67-positive cells correspond to greater malignancy.

Introduction 11

Monoclonal antibody MIB-1 is used to recognize this antigen, and so the usefulness of thisantibody is discussed in detail in Chapter 2. Immunohistochemical methods usingmicrowave heating or autoclave treatment for localizing Ki-67 are presented.

Proliferating cell nuclear antigen (PCNA) is an auxiliary protein to DNA polymerase8 and is intimately associated with DNA replication. Indeed, direct interaction betweenDNA polymerase and its processivity factor PCNA is essential for effective replication ofthe eukaryotic genome. This protein also plays a key role in other functions, such asnucleotide excision repair, mismatch repair, base excision repair, cell cycle control, apop-tosis, and transcription. These interactions support the concept that PCNA plays a centralrole in connecting all these important cellular processes and can function as cellular com-municator in cells. Clinically useful activity of PCNA can be identified by immunohisto-chemistry and flow cytometry. Expression of this antigen in a cell population equates tothe growth fraction, that is, the proportion of cells involved in an active cell cycle. BecausePCNA is expressed in all cycling cells, the entire proportion of dividing cells present at anyinstant in a population can be detected. Details of immunohistochemical staining of PCNAon cryostat and paraffin-embedded sections are presented in this volume.

In recent years evidence has increasingly demonstrated the importance of proto-oncogenes in the pathogenesis of diseases, including breast cancer. Amplification ofHER-2/neu gene is found in ~25% of human breast cancers and results in the overex-pression of p185 oncoprotein. Amplification of the gene in breast cancer patients is corre-lated with shorter disease-free states and poorer overall survival rates than in patientsshowing no such amplification. Accurate detection of the gene amplification in breast can-cer tissues is important in determining patient prognosis as well as response to standardchemotherapeutic agents. Moreover, it is currently the sole criterion for selecting patientsfor HER-2/neu-targeted therapy with the recombinant humanized anti-p185 antibodyHerceptin (trastuzumab) (Pauletti et al., 2000). Overexpression of this protein in breastcancer is associated with adverse prognostic factors that include advanced pathologicalstage, number of metastatic axillary lymph nodes, absence of estrogen and progesteronereceptors, increased S-phase fraction, DNA ploidy, and high nuclear grade.

Because the gene product is ultimately responsible for the biological activity of thegene, it is apparent that direct measurement of the protein or immunohistochemical analysisis as clinically relevant as is the determination of the number of gene copies (Battifora et al.,1991). Immunohistochemistry is superior to biochemical assays because it eliminates thedilution effect caused by variable amounts of stroma and other nonneoplastic tissues.Another advantage of immunohistochemistry is its ability to detect overproduction of anoncoprotein resulting from a mechanism other than gene amplification. Amplification ofHER-2/neu gene and Overexpression of p185 are also found in many tissues other than thosewith breast cancer. Immunohistochemical detection of p185 is presented in Chapter 12.

CYTOGENETIC EVALUATION OF TUMORS

A brief discussion on the usefulness of cytogenetic evaluation of tumors is in order.The diagnosis of tumors on the basis of information obtained from immunohistochemicalstaining alone on occasion may pose a challenge to the pathologist. Cytogenetics, whichis another approach to determine the histogenetic origin of some tumors and to identify

12 Chapter 1

sites of gene deregulation for molecular analysis, can provide an important adjunct todiagnostic surgical pathology. For example, karyotypic analyses are helpful in the differ-ential diagnosis of histologically similar small round cell tumors, including lymphoma andneuroblastoma (Sreekantaiah et al., 1994). These tumors are composed of primitive cellsthat often lack distinguishing features.

Each of these tumors contains specific chromosome changes; thus, cytogenetic analy-sis provides a reliable approach that can distinguish between these neoplasms. In addition,the identification of diagnostic chromosome translocations in histologically undifferenti-ated tumors may support a diagnosis that is doubtful on histological grounds alone or mayeven lead to a reconsideration of the histological diagnosis. This approach can aid indirecting therapy, determining prognosis, and identifying sites of gene perturbation formolecular characterization.

Unfortunately, cytogenetic evaluation of tumors is still a relatively underutilizedapproach. However, new potentially promising tumor markers have been introduced basedon the molecular genetic cancer research. Various genetic alterations important in carcino-genesis, of which alterations in the ras oncogenes and the p53 tumor suppressor gene are themost common, have now been described. Both of these are useful targets for diagnostic pur-poses. This is substantiated by considering that p53 alterations are among the most frequentgenetic alterations in human malignancies. Similarly, clinical application of ras gene muta-tion, for example in the diagnosis of pancreatic adenocarcinoma, has been well established(e.g., Berthelemy et al., 1995). Chromosomal abnormalities in many tumors and their diag-nostic relevance are discussed by Sreekantaiah et al. (1994) and Gisselsson et al. (2001).

Finally, cancer is a genetic disease, for acquired genetic aberrations cause the disease.Changes in antigen expression detected with immunohistochemistry in some instancesreflects genetic alterations. The detection of these aberrations at the chromosome and genelevels improved diagnosis, prognosis, and therapy. Therefore, the combination of morphol-ogy with genetics is a major step toward a better understanding of human disease. A num-ber of techniques that facilitate this combination are available, such as in situ hybridization,comparative genomic hybridization, expression profiling using array technologies, highthroughput screening approaches, and phenotype/genotype correlations on the DNA,RNA, or protein level (Ried et al., 1999). Technological innovations such as image analy-sis systems, cytophotometric and integrated densitometric quantitation, and computerhard- and software development also assist in this effort.

GENETIC INSTABILITY OF TUMORS

It is well established that cancer is caused by the accumulation of mutations in thegenes that are directly responsible for cell birth or cell death. Genetic instabilities are aconsequence of cancer mutations. One or more mutations initiate tumor growth, whichgive the tumorous cell a selective advantage over other cells. The clone derived from thetumorous cell then expands. Successive mutations occur, each followed by waves of clonalexpansion. Information on the molecular and physiological bases of genetic instability oftumors is resulting in new approaches to treating cancer.

One of two levels of genetic instability correlates with the vast majority of cancers.In most cancers genetic instability is observed at the chromosome level, resulting in losses

Introduction 13

or gains of whole chromosomes or large portions of them (Lengauer et al., 1998). On theother hand, in a small subset of tumors, genetic instability is observed at the nucleotidelevel and results in base substitutions, deletions, or insertions of a few nucleotides. Anunderstanding of these instabilities is providing new insights into tumor pathogenesis.

Four major types of genetic alterations that affect growth-controlling genes have beenidentified in neoplastic cells and are the basis of human cancers.

1.

2.

3.

4.

Sequence changes involving base substitutions, deletions, or insertions of a fewnucleotides. This type of subtle change is exemplified by missense mutations inthe K-ras gene, which occur in more than 80% of pancreatic cancers (Almogueraet al., 1988). These changes cannot be detected with cytogenetic analysis.Alterations in chromosome number involve losses or gains of whole chromosomesand are found in almost all major types of human tumors. Losses of heterozygosity(losses of a maternal or paternal allele) are widespread. The average cancer of thecolon, breast, pancreas, or prostate may lose ~25% of its alleles, and some tumorsmay lose more than half of their alleles (e.g., Vogelstein et al., 1989). Such can-cers exhibit a true chromosomal instability that persists throughout the lifetime ofthe tumor. It is known that chromosome 10 is lost in glioblastomas, inactivatingthe tumor suppressor gene PTEN (Wang et al., 1997). The gain of chromosome 7in papillary renal carcinomas indicates a duplication of a mutant MET oncogene(Zhuang et al., 1998).Chromosome translocations are common in certain human cancers. Translocationssuch as fusions of different chromosomes or of normally noncontiguous segments ofa single chromosome can be detected cytogenetically. At the molecular level, suchtranslocations can produce fusions between two different genes, imparting to thefused transcript the tumorigenic properties. For example, in chronic myelogenousleukemias the carboxy terminus of the c-abl gene on chromosome 9 is joined to theamino terminus of the BCR gene on chromosome 22 (Nowell, 1997).

Translocation can also cause gains or losses of chromosomal material andgenerate new gene products. Simple translocations are characterized by distinctiverearrangements of chromosomal segments in specific neoplastic diseases, includ-ing leukemias and lymphomas. These specific translocations are necessary for thedevelopment and progression of the neoplasms in which they occur.Gene amplification is an important process in human cancers, as it is associatedwith tumor progression, has prognostic significance, and provides a target for ther-apeutics (Lengauer et al., 1998); an example is the amplification of HER-2/neu inbreast cancers. At the cytogenetic level, gene amplifications can be detected ashomogeneously stained regions or double minutes. At the molecular level, multiplecopies of an amplicon containing a growth-promoting gene can be detected. Theamplification of N-myc oncogene that occurs in about one-third of advanced neu-roblastomas is a good example of tumor progression (Seeger et al., 1985).

Almost all solid tumors are genetically unstable. Translocations and gene amplifica-tions add to the chromosomal abnormalities and may reflect additional mechanismsfor generating genetic instability that occurs as tumors grow. Genetic instability is thecause of both tumor progression and tumor heterogeneity. As a result, no two tumors areexactly alike and no single tumor is constituted of genetically identical cells. Tumor

14 Chapter 1

heterogeneity is an obstacle in standardizing the diagnosis, as well as in selecting thera-peutic strategies. However, it should be noted that although genetic instability is essentialfor neoplasia to develop, the instability may provide equally valid therapeutic targets.

TUMOR HETEROGENEITY

Heterogeneity has been reported in a variety of human tumors. Intraindividual hetero-geneity is defined as subpopulations of tumor cells found within one tumor. Heterogeneityamong different tumors is called interindividual heterogeneity. The detection of hetero-geneity at the tumor level is relevant to explanations of clonality differences, metastaticpotential of human tumors, and response to therapy under different treatment regimes. Theexistence of heterogeneity can be explained by genetic instability of malignant progenitoror stem cells. In addition to other influences, heterogeneity might be one individual factorthat explains differences in response and outcome of patients under treatment.

Heterogeneity at the tumor cell level can be detected by histological, immunohisto-chemical, molecular genetic and flow cytometric methods, and polymerase chain reaction.Recently, multiparameter flow cytometry was used for detecting tumor cell heterogeneity(Könemann et al., 2000). This immunophenotyping, with its advantages of characterizingsimultaneously a variety of different antigens, allows detection of interindividual as wellas intraindividual heterogeneity and malignant subpopulations. The method provides thepossibility of characterizing solid tumors according to their immunophenotype and DNAcontent. Molecules that are potentially involved in tumor invasion, metastasis, differentia-tion/maturation, and cell interactions can be chosen as target antigens. These include adhe-sion molecules, cell activation antigens, and cytokine and growth factor receptors.

Histological Microdissection

Tissue heterogeneity of histological specimens is well known. Not only neoplastic cellsare heterogenous; a tumor may contain a variable admixture of stromal cells, inflammatoryinfiltrates, endothelial cells, and preexisting tissue. This complexity hinders the study ofmolecular genetic alterations. Such studies require precise correlation of molecular geneticcharacteristics to well-defined cell populations. The presence of multiple cell types close toone another in the tumor may limit the precise significance of changes in specific cells. As aresult, even sophisticated techniques become less useful when applied to bulk tissue.

Study of uniform cell populations is a prerequisite to understanding differential geneexpression in tumors. A number of mechanical techniques for microdissection have beendeveloped to isolate cells for analysis from histological sections (Turbett et al., 1996;Youngson et al., 1995; Going and Lamb, 1996; Moskaluk and Kern, 1997; Lee et al., 1988;Zhuang et al., 1995). The most sophisticated technique is laser-assisted and suitable formicrodissection of single cells with minimal risk of contamination (Becker et al., 1997).Some of the microdissection methods are satisfactory but also have certain disadvantages;for example, laser-assisted techniques require expensive equipment.

To overcome some of the limitations of microdissection methods presently inuse, recently a mechanical technique was developed by Harsch et al. (2001). This device

Introduction 15

consists of an ultrasonically oscillating needle and a piezo-driven micropipette for rapidand precise histological microdissection. The oscillating needle is used to fragment the tis-sue into subcellular particles that are aspirated into the pipette tip. The method can beapplied to paraffin sections or unfixed cryostat sections. It allows a sharp demarcationbetween the dissected area and unwanted tissue that remains intact for further analysis.Individual colonic crypts can be dissected without collecting any adjacent stroma. Thus,the technique is useful for determining gene expression in defined cell populations.

ANTITUMOR VACCINES

Advances in the molecular characterization of human tumors have led to a betterunderstanding of tumor immunology. These advances reveal that cancer cells exhibitspecific patterns of gene expression or molecular alterations, compared with normalcellular counterparts, resulting in the production of tumor-associated antigens. A numberof such antigens are self-antigens, which allow conceiving and designing of specific vac-cines against virtually every solid tumor. Thus, efficient cancer vaccines should be able toneutralize immune tolerance against such antigens.

The idea of controlling cancer by stimulating the immune system is not a recent one.In fact, more than a century ago, bacterial extracts were used for stimulating tumor-specificimmune responses (Coley, 1893). Subsequently, immunostimulatory cytokines were usedagainst a number of cancers (Marincola et al., 1995). Passive transfer of cytotoxic immunecells (e.g., lymphocytes) was also tested in humans (Yee et al., 1997). Recently, a numberof studies indicate that therapeutic vaccines might be useful in restoring immune defensesagainst cancer. The following discussion summarizes possible uses of cancer vaccines.

An important future strategy for cancer immunotherapy is the use of the next genera-tion of antigen-specific cancer vaccines. Until now, most clinical trials have been performedwith end-stage cancer patients because data on vaccine-induced immune responses are lim-ited. There are two variations of the development of antitumor vaccination strategies: (1)developing vaccines utilizing whole tumor cells and (2) working on vaccines targetingdefined antigens. The advantage of tumor cell–based vaccines is that these in principle com-prise all relevant tumor antigens. Consequently, there is no need for prior identification ofthe tumor antigens to be included in the vaccine. The limitation of this approach is that it isvery difficult to understand the therapeutic effect of these vaccines on the disease.

In contrast, the use of vaccines comprising defined antigens enables the improvementof vaccine strategies based on empirical findings (Offringa et al., 2000). This approachallows the systematic analysis of vaccine-induced immunity in relation to clinical response.This advantage strongly argues for the usefulness of antigen-specific anticancer vaccines.

The protective effect of tumor cell vaccination is thought to involve defined T cellresponses. However, only in selected cases does the detection of T cell immunity againstdefined antigens coincide with clinical response (Sun et al., 1999). For example, vaccinationof patients exhibiting residual B cell lymphoma, using the tumor-specific immunoglobulinidiotype as an antigen, was shown to result in sustained molecular remission, accompaniedby idiotype-specific T cell and antibody responses (Reichardt et al, 1999). Another positiveexample demonstrates that a human papillomavirus-specific vaccine can have therapeuticefficacy against benign neoplastic tumors such as genital warts (Lacey et al., 1999).

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Presently, vaccines expressing tumor-associated antigens are being tested in a thera-peutic setting. A specific example of a candidate vaccine is Gastrimmune, which is beingtested in patients with advanced gastric and pancreatic cancers (Greten and Jaffe, 1999).The aim of such testing is to specifically evoke or restimulate a specific antitumor immuneeffector response. The likely immune effector mechanisms involved in tumor control/elim-ination consist of innate immunity, cytotoxic T cells, NK cells, and antibodies. In addition,the activation of specific T helper cells is thought to be important not only to orient andregulate the immune response but also to sustain immune effector mechanisms in vivo(Bonnet et al., 2001).

One way to use vaccines against cancer is by preventing infection by pathogensknown to predispose to certain cancers. Approximately 16% of the worldwide incidenceof cancer can be attributed to infectious pathogens (Ames et al., 1995). The objective is todecrease cancer incidence by using vaccines against the pathogens. A well-known recentexample of this approach is the nationwide hepatitis B vaccination program in Taiwan.This resulted in the substantial decline in the incidence of hepatocellular carcinoma in chil-dren (Chang et al., 1997). Other cancers caused or facilitated by viruses against whichexperimental vaccines are available include Burkitt lymphoma, nasopharynx cancer, adultT cell leukemia, cervical carcinoma, B cell gastric lymphoma, and gastric carcinoma.

The efficacy of DNA vaccines has also been compared with that of protein subunitvaccines. The use of plasmid DNAs as vaccines has several potential advantages in addi-tion to ease of manipulation and preparation. For example, unlike most protein subunitvaccines, DNA vaccines are potentially able to stimulate both cell-mediated and humoralimmunity. On the other hand, both subunit vaccines and DNA vaccines have perceivedsafety advantages over the use of live virus vaccines. Recently, Nass et al. (2001) haveshown that a DNA vaccine expressing a single herpes simplex virus glycoprotein is saferthan live virus immunization in immunocompromised animals and that the magnitude ofprotection in immuncompetent animals against subsequent challenge approaches thestrength of protection achieved by sublethal infection.

Another recent example of the development of a DNA vaccine is against theHER-2/neu expressing carcinomas. Foy et al. (2001) have utilized an in vivo murine tumorexpressing human HER-2/neu for evaluating potential HER-2/neu vaccines consisting offull-length or various subunits of HER-2/neu delivered in protein or plasmid DNA form.This study demonstrates that protective immunity against HER-2/neu–expressing tumorchallenge can be achieved by these vaccines. Partial protective immunity is also observedfollowing vaccination with the intracellular domain (ICD), but not extracellular domain(ECD), protein subunit of HER-2/neu. The mechanism of protection elicited by plasmidDNA vaccination is thought to be exclusively CD4-dependent, whereas the protection withICD protein vaccination requires both CD4 and CD8 T cells. These early studies indicatethat multiple forms of HER-2/neu vaccines would be more effective in eliciting the pro-tective HER-2/neu–specific antitumor responses.

It is expected that in the near future antigen-specific vaccines will be applied effec-tively to induce strong T cell immune responses in patients displaying less progressedstages of disease. A comprehensive discussion on the development of therapeutic cancervaccines (molecular vaccines) has been presented by Moingeon (2001) and Monzavi-Karbassi and Kieber-Emmons (2001).

Introduction 17

MOLECULAR GENETICS

Basic mechanisms of pathogenesis can be elucidated through molecular geneticresearch. Based on such information, specificity, sensitivity, and efficiency of diagnosticand prognostic tests for many diseases can be improved. As a result, new insights intotherapeutic approaches can be developed, and their effectiveness can be assessed morereliably. The Human Genome Project has focused attention on the association of mutationsin nuclear DNA with human diseases. A complete working draft of the human DNAsequence was completed in spring 2000. This project continues to define disease-associatedmutations, and the number of clinically useful molecular pathological techniques andassays has also been increasing.

The technology is available to extract and amplify DNA from minuscule archival andfresh samples as diverse as blood, urine, sputum, and solid tissues, including fixed andparaffin-embedded tissues. Also, DNA can be purified even from dried blood spots foramplification and mutation analysis (Kiechle, 1999), thus permitting the study of an indi-vidual’s inherited genes and mutations. Hereditary information can also be obtained byassessing RNA, proteins, and enzyme activities. Such assessments can be morphological,immunological, or biochemical. The following examples indicate the usefulness of molec-ular pathology in better understanding diseases.

The usefulness of identifying molecular alterations underlying neoplasia is obviousin borderline tumors. Whether such tumors should be classified as benign or malignant,and whether they represent a precursor of frank malignancy, is a matter of controversydespite extensive clinical and pathological studies. In many cases this problem can besolved by using the biology of tumors as the genetic indicator of malignancy. Thisapproach is exemplified by mutations in the p53 tumor suppressor gene and Ki-ras onco-gene, which are the most common genetic alterations in human cancers. These mutationsare used as genetic indicators of malignancy.

Methods are available to analyze abnormalities in these genes using paraffin sectionsof neoplasms (Frank et al., 1994; Caduff et al., 1995). Mutations in codon 12 of Ki-ras canbe identified in DNA extracted from paraffin sections using an amplified created restric-tion site method, followed by confirmation using gene sequencing (Lin et al., 1993).Missense mutations in the p53 gene can be identified with an immunohistochemical sur-rogate to detect the nuclear accumulation of p53 protein that results from such mutations(Kerns et al., 1992).

Recently, Caduff et al. (1999) have evaluated abnormalities in p53 and Ki-ras in malig-nant and borderline ovarian tumors of various histological types in paraffin-embedded tis-sues. The patterns of these genetic alterations in borderline and malignant neoplasms werecompared and correlated with cell type and stage. This preliminary molecular analysis sug-gests that serous borderline tumors have the same molecular features usually associatedwith malignancy but are unlikely to represent a precursor of invasive serous carcinoma.On the other hand, mucinous borderline tumors may represent a precursor or variant ofmucinous carcinoma of the ovary.

Another example is the study of microsatellites that are short DNA loci having sim-ple sequence repeats that are widely distributed throughout the human genome. They area valuable source for human genetic linkage analysis and molecular cancer research

18 Chapter 1

because of allelic polymorphisms. In hereditary nonpolyposis colorectal cancer, an auto-somal-dominant disorder accounting for 2–10% of all colorectal cancers, length alterationsin single mononucleotide or dinucleotide repeats (microsatellite instability [MSI]) occur(Raedle et al., 1999).

The MSI is used as a diagnostic criterion of replication errors caused by various muta-tions in at least five mismatch repair genes (Lynch and Smyrk, 1996). Therefore, MSIanalysis is useful in clinical practice to identify patients with hereditary nonpolyposis col-orectal cancer. Raedle et al. (1999) have presented a rapid DNA extraction method (rapidmicrosatellite analysis) for analyzing replication errors in paraffin-embedded tissues.

Southern blotting and polymerase chain reaction are being used for detecting B- andT-cell clonality in lymphoproliferative diseases, including mantle cell lymphoma and lym-phoma of the breast (Medeiros and Carr, 1999). Molecular genetic tests are currentlyimportant ancillary tools for the diagnosis and classification of malignancy, and their roleis likely to increase in the future.

The positive aspects of molecular genetics and molecular pathology mentioned aboveneed to be balanced with ethical concerns to safeguard the rights and welfare of humansubjects (Sobel, 1999). The state and federal regulations protecting patient’s privacy andwelfare must be observed.

cDNA MICROARRAY TECHNOLOGY

In conjunction with detailed understanding of the human genome, sophisticated meth-ods are required for gene expression analysis and gene discovery. These approaches will pro-vide insights into growth, development, differentiation, homeostasis, aging, and disease onset.One such recently introduced method is cDNA microarray or DNA-chip technology, whichfacilitates monitoring the expression of hundreds and thousands of genes simultaneously andprovides a format for identifying genes as well as alterations in their activity (Kononen et al.,1998). Because of the wide spectrum of genes and endogenous mediators involved, this tech-nology is helpful in recognizing chronic diseases. As the cDNA microarray technique allowslarge-scale expression analysis, it is well suited to observe the broad effects of oncogenic tran-scription factors on gene expression and potentially clarify their role in oncogenesis.

The cDNA technology uses cDNA sequences or cDNA inserts of a library for poly-merase chain reaction (PCR) amplification, which are arrayed on a glass slide with high-speed robotics at a density of 1,000 cDNA sequences per square centimeter. In otherwords, microarrays can be constructed from specific cDNA clones of interest, a cDNAlibrary, or a selected number of open reading frames from a genome sequencing databaseto allow a large-scale functional analysis of expressed sequences. These microarrays serveas gene targets for hybridization to cDNA probes prepared from RNA samples of cells andtissues. A two-color fluorescence labeling technique can be used to prepare the cDNAprobes so that a simultaneous hybridization, but separate detection, of signals provides thecomparative analysis and the relative abundance of specific genes expressed.

The cDNA technology is essentially an array-based, high-throughput protocol thatdetermines gene expression and copy number survey of very large numbers of tumors. Asmany as 1,000 cylindrical tissue biopsies from individual tumors can be distributed in asingle tissue microarray. Sections of the microarray also provide targets for parallel in situ

Introduction 19

detection of DNA, RNA, and protein targets in each specimen on the array. Moreover, con-secutive sections allow rapid analysis of hundreds of molecular markers in the same set ofspecimens. Another advantage is that sufficient cDNA for hybridization to a microarraycan be produced from as little as 1 mg of tissue.

This technology can be used to profile complex diseases and discover novel disease-related genes. It can dissect complex human diseases by analyzing the pattern of geneexpression. The cDNA microarray method could provide new targets for drug develop-ment and disease therapies and thus facilitate improved treatment of chronic diseases thatare challenging because of their complexity. Listed below are examples of diseases whosemolecular characteristics have been determined using gene arrays.

Ljubimova et al. (2001) have used 11,000 gene microarrays for identifying geneexpression profiles in brain tumors, including high-grade gliomas (glioblastoma multiforme[GBM] and anaplastic astrocytoma), low-grade astrocytomas, and benign extraaxial braintumors (meningioma), and then compared them with normal brain tissue. In this study thegene array method was combined with reverse transcriptase (RT)-PCR and immunohisto-chemical evaluation of glial tumors. All GBMs overexpressed 14 known genes, whereasthese genes were barely detectable in normal human brain tissue. This study also showed thatlaminin-80–containing GBMs recurred significantly sooner after surgical removal than didGBMs with a predominant expression of laminin-9. Thus, overexpression of laminin-8 intumor blood vessel walls may be an indicator of time to recurrence for patients with GBM.

Another example of the involvement of many different genes in a cancer is renal cellcarcinoma. This cancer is one of the 10 most frequent malignancies in western countries.Genes involved in the initiation and progression of this cancer include the vonHippel–Lindau gene on chromosome 3p, the epidermal growth factor receptor gene onchromosome 7p, the transforming growth factor gene on chromosome 2p, and the c-myconcogene on chromosome 8q (Siezinger et al., 1988; Moch et al., 1998; Lager et al., 1994;Yao et al., 1998). Other genes involved in renal cancer are currently not known.

Moch et al. (1999) have combined tumor arrays and cDNA arrays for rapid identifi-cation of genes and their role in renal cell carcinoma. They constructed a kidney cancertissue array consisting of 532 renal tumors, 386 of which had clinical follow-up data avail-able. There were 89 differentially expressed genes in the cancer cell line CRL-1933, oneof them encoding for vimentin. Vimentin expression was significantly associated with poorpatient prognosis independent of grade or stage.

cDNA microarray technology has also been used for verifying the involvement of anumber of genes in another complex disease, rheumatoid arthritis (Heller et al., 1997). Inthis disease inflammation of the joint is caused by the gene products of many different celltypes present in the synovium and cartilage tissues plus those infiltrating from the circu-lating blood. In this study the presence of gene products, such as matrix degrading metal-loproteinase (MMP), macrophage inflammatory protein (MIP), and human matrixmetalloelastase (HME), was verified. The expression profiles of the genes demonstrate theutility of the microarrays in determining the hierarachy of signaling events.

The downstream effects of both PAX3 and PAX3-FKHR on NIH 3T3 cells withcDNA microarrays has also been monitored (Khan et al., 1999). This study elucidated thepattern of gene expression induced by these two oncogenic transcription factors in thesecells; these factors showed significant myogenic properties. Other recent examples of theapplication of gene arrays for determining the molecular parameters of individual tumors

20 Chapter 1

are ovarian and cervical cancers and metastatic versus primary breast cancer (Ono et al.,2000; Shim et al., 1998; Nacht et al., 1999). New subclasses of leukemia have also beenidentified using gene arrays, which have become critical for the successful treatment ofpatients (Golub et al., 1999).

Because the individual arrayed tissue samples are very small (0.6 mm in diameter),one might ask if these specimens are representative of their donor tumors! To answer thisquestion, Nocito et al. (2001) studied a set of 2,317 bladder tumors that had been previouslyanalyzed for histological grade and Ki-67 labeling index. The histological grade and theKi-67 labeling index were determined for every arrayed tumor sample. The grade andKi-67 information obtained on minute arrayed samples were highly similar to the dataobtained on large sections. On the basis of this evidence, it can be stated that intratumor het-erogeneity does not significantly affect the ability to detect clinicopathological correlationson the tissue microarrays. It is concluded that tissue microarray is an important tool forrapid identification of biological or clinically significant molecular alterations in tumors.

ANGIOGENESIS

Two distinct processes, vasculogenesis and angiogenesis, form blood vessels.Vasculogenesis is responsible for the de novo differentiation of endothelial cells frommesodermal precursors and occurs during embryonic development, leading to the forma-tion of a primary vascular plexus. Angiogenesis, on the other hand, is the process of sprout-ing and configuring new blood vessels from preexisting ones. It is a complexphenomenon comprising a series of cellular events that lead to the neovascularization asso-ciated with the process of tumor growth, metastasis, inflammation, and wound healing(Fig. 1.5/Plate 1A). Angiogenesis that occurs in wound repair and formation of collateralblood vessels following an infarct, ischemia, or reduced blood flow is advantageous for

Introduction 21

normal tissue function. It should be noted that tumor angiogenesis is not sufficient to causetumor spread and patient death. Tumor cells must also proliferate, penetrate host tissuesand vessels, survive within the vasculature, escape the host immune system, and then begingrowth at a new body site (Weidner, 1998). Before discussing the role of angiogenesis indisease, it is relevant to explain the process of angiogenesis.

The complex process of angiogenesis includes the recruitment of nearby endothelialcells, their activation, degradation of the vascular basement membrane, proliferation and

form a new capillary (Albini et al., 2000). Tumor-induced endothelial cell activation leads tothe acquisition of a phenotype characterized by chemotactic motility, basement membraneinvasion, and proliferation. These events are followed by differentiation into a new vessel.

During the last decade there have been significant advances in the understanding offunctional mechanisms of the molecules involved in angiogenesis. Angiogenesis is medi-ated by multiple positive and negative regulator molecules released by tumor cells, intratu-moral macrophages, mast cells, and endothelial cells. The balance of the effects of thesemediators determines the outcome of this process. At least three groups of extracellular sig-nals are involved in angiogenesis: (1) soluble growth molecules such as acid and basicfibroblast growth factors and vascular endothelial growth factor (discussed later) that affectendothelial cell growth and differentiation; (2) factors such as transforming growth factorand angiogenin that inhibit proliferation and enhance differentiation of endothelial cells; (3)extracellular matrix–bound cytokines released by proteolysis, which contribute to angio-genic regulation. Other growth factors implicated in different steps of angiogenesis areplatelet-derived growth factor, hepatocyte growth factor, and angiopoietins 1 and 2. Also,various endothelial surface molecules, such as CD31, CD144, and integrins, play arole in angiogenesis.

Some of the above-mentioned secreted factors are angiogenic, whereas others areangiostatic. Thus, angiogenesis is mediated by multiple positive and negative regulatorymolecules released by both tumor cells and the surrounding normal cells. The balancebetween these regulators determines whether or not neovascularization will occur. Indeed,antiangiogenic therapy is based on the use of negative regulators of neovascularizationaimed at suppressing the proangiogenic signal or increasing the inhibitory signals. Albiniet al. (2000) have used the gene therapy approach using class I interferons for effectivelyinhibiting tumor angiogenesis and growth of vascular tumors.

Although overwhelming evidence indicates that endothelial cells are central to theangiogenic process, the following discussion proposes the role of tumors in the formationof blood vessels. According to Maniotis et al. (1999) and Folberg et al. (2000), blood ves-sels of malignant eye tumors known as uveal melanomas are formed by tumor cells insteadof endothelial cells. These highly aggressive and metastatic cells are capable of forming invivo and in vitro vascular channels, which consist of a basement membrane that stains pos-itive with the periodic acid–Schiff (PAS) reagent in the absence of endothelial cells andfibroblasts. The generation of such channels is termed vasculogenic mimicry. This evidencesuggests that angiogenesis may not be the only mechanism responsible for creating tumormicrocirculation. Therefore, methods used to identify the tumor microcirculation by stain-ing endothelial cells may not be applicable to tumors that express vasculogenic mimicry.

However, certain aspects of the concept of vasculogenic mimicry have beenquestioned by McDonald et al. (2000). They indicate that PAS-stained channels do not

22 Chapter 1

represent the microvascular architecture, and endothelial cell–lined blood vessels are alsopresent in uveal melanomas. They moreover report that tumor cell-lined vessels are infre-quent in these melanomas. Nevertheless, tumor cells can acquire a new phenotype and par-ticipate in the formation of blood vessels. It is concluded that the extent and thepathophysiological significance of cancer cells becoming lining cells and participating inthe formation of blood vessels in tumor is still unclear.

Information on the angiogenesis regulatory molecules has produced new therapeuticstrategies for suppressing angiogenesis and tumor growth or promoting angiogenesis againstcoronary and peripheral ischemia and stimulation of wound healing (Thompsonet al., 1999; Kahn et al., 2000). Limited space does not allow discussion of these aspects ofangiogenesis, except for VEGF, which is the most potent angiogenic factor (see pages 23–24).

Angiogenesis plays an important role in the development and progression of a num-ber of disease states, including various cancers, diabetic retinopathy, macular degenera-tion, psoriasis, and rheumatic arthritis. The tumor microcirculation plays a key role inhematogenous dissemination of cancers. There is compelling evidence that angiogenesisis indeed critical for tumor growth progression and metastasis because tumors require newblood vessels to achieve a size larger than 2–3 mm. Also, a considerable amount of evi-dence suggests that tumor angiogenesis is crucial for the growth of solid tumors in vivo(Folkman, 1996). The growth of tumors (including solid tumors) must be preceded by anincrease in capillaries and newly formed blood vessels that provide tumor cells with oxy-gen and nutrients as well as paracrine mediators. The blood vessels also remove wasteproducts.

A large number of tumor blood vessels increases the opportunity of the tumor cells toenter the circulation. In fact, the newly formed capillaries usually have a fragmented base-ment membrane, facilitating easier invasion. In the prevascular phase, with little or noangiogenic activity, the tumor is unable to expand beyond a few cubic millimeters, butonce angiogenic factors are released in sufficient number, the onset of angiogenic activitystimulates rapid expansion of the tumor.

The microvessel density of the tumor mass, a measure of tumor angiogenesis,correlates with metastasis and can be used as an independent prognostic factor in themanagement of cancer (Jacquemier et al., 1998). A number of studies indicate thatmicrovessel density gives prognostic information on breast cancer (Weidner et al., 1993).With respect to prognostic carcinoma, it is thought that a low vascular density correlateswith significantly longer survival duration than with carcinomas having high vascular den-sity. Thus, neovascularization has proven to be an independent predictor of pathologicalstate in prostatic carcinoma. A correlation between the endocrine differentiation andincreased neovascularization in prostatic cancer has also been reported (Grobholz et al.,2000). High-grade tumors with a high neuroendocrine differentiation and increased neo-vascularization indicate high risk and unfavorable outcome.

Although the role of angiogenesis as a prognostic factor has been most widelyanalyzed in breast cancer, angiogenesis also plays an important prognostic role in othercarcinomas such as gastric cancer. This cancer is a highly aggressive malignancy with poorprognosis and low survival rates. Sanz-Ortega et al. (2000) have evaluated advanced gas-tric cancers for the expression of oncogenes HER-2/neu (c-erbB-2), c-myc, and epidermalgrowth factor receptor, as well as microvessel density. Avidin-biotin immunohistochem-istry using CD34 stained paraffin sections has shown that tumor angiogenesis is the mostimportant independent prognostic indicator to predict overall survival.

Introduction 23

Immunohistochemistry using primary antibody against rabbit antihuman VEGF(diluted 1:1000) has demonstrated that angiogenesis is also a vital process in cartilaginoustumors and that VEGF expression by malignant chondrocytes is required for the formationof intracartilage vessels (Ayala et al., 2000). Intracartilage vessels might be involved in theacquisition of metastatic potential by cartilage tumors.

Squamous cell carcinoma is also characterized by a richly vascularized stroma andoverexpression of VEGF. This carcinoma of the skin is a malignant tumor of epidermalkeratinocytes with a destructive growth pattern, and it has the ability to metastasize. It hasbeen demonstrated that selective overexpression of VEGF in highly differentiated squa-mous cell carcinomas is sufficient to induce tumor invasiveness as well as to promotetumor growth and angiogenesis (Detmar et al., 2000). The tumor stroma also plays anactive role in the progression of this cancer.

As stated earlier, angiogenesis plays a role in repairing blood vessel injury. Two sys-tems, angiogenesis and hemostasis, remain poised for repair of blood vessel injury. At thesite of blood vessel injury, adhered platelets secrete both positive and negative regulatorsof angiogenesis, mainly from internal The positive regulators include VEGF;negative regulators include platelet factor 4. Hepatocyte growth factor affects both stimu-lation and suppression of angiogenesis. On the other hand, the hemostatic system main-tains the liquid flow of blood by regulating platelet adherence and fibrin deposition.Browder et al. (2000) have discussed in detail how angiogenesis is coordinated by and withhemostasis during blood vessel repair.

In conclusion, sufficient evidence is available indicating that assessment of microves-sel density is very useful in tumor biology. However, consensus on the prognostic value ofangiogenesis is lacking. The main reasons for conflicting results consist of the study of dif-ferent angiogenic-regulating factors, the use of varying methodologies for measuringmicrovessel density, and the significant intraobserver variation that exists in interpretationof the number of positive vessels and the optimal way in which fields are selected, that is,hot spot (the most vascular area of the tumor) versus general counting of vessels. These rea-sons do not allow meaningful comparison among the results reported in various studies.

Standardization of processing conditions, such as tissue section preparation, staining,careful selection of the hot spot, and a strict protocol for defining microvessels, canachieve adequate reproducibility. However, despite these precautions, manual counting ofmicrovessels and selection of the hot spot are still subjective and therefore not always fullyreproducible. These problems can be significantly minimized by using fully automatedmicrovessel counting and hot spot selection by image processing of whole tumor sections,for example, in invasive breast cancer (Beliën et al., 1999). In comparison with the man-ual method, the automated procedure reduces the microvessel measurement time when thecomplete tumor is scanned, achieves greater accuracy and objectivity of hot spot selection,and allows visual inspection and relocation of each measurement field awards. The reasonsfor contradictory interpretations and potential remedies have been presented in more detailby Hansen et al. (1998).

Vascular Endothelial Growth Factor

The vascular endothelial growth factor (VEGF) is a member of the six-member VEGFfamily: VEGF, placenta growth factor, VEGF-B, VEGF-C, VEGF-D, and VEGF-E. These

24 Chapter 1

members have overlapping but specific roles in the growth of new blood vessels. The fol-lowing discussion is limited to only one member, VEGF (also known as vascular perme-ability factor), which is the most important and most frequently studied angiogenic factor.It is a homodimeric 34–42 kDa glycosylated heparin-binding glycoprotein. Alternativeexon splicing of the VEGF gene produces multiple species of mRNA, which encode dif-ferent VEGF protein isoforms having subunit polypeptides of 121, 145, 165, 189, or 206amino acid residues (Neufeld et al., 1999).

Vascular endothelial growth factor has three receptors [VEGFR-1 (Flt-1), VEGFR-2(KDR/Flk-1), and VEGFR-3 (FLT-4)], each consisting of seven immunoglobulin-homologydomains, a transmembrane sequence, and an intracellular portion containing a split kinasedomain (Shibuya, 1995). Ligand (VEGF) binding induces receptor dimerization and sub-sequent auto/transphosphorylation. The receptors have distinct roles in vasculogenesis andangiogenesis during embryonic development. Precise roles of the three receptors havebeen discussed by Veikkola and Alitalo (1999).

Transcription of VEGF mRNA is induced by a variety of growth factors and cytokines,including platelet-derived growth factor-BB, epidermal growth factor, tumor necrosis

transforming growth and (Ferrara and Davis-Smyth,1997). Tissue oxygen tension tightly regulates VEGF levels, and exposure to hypoxiarapidly and reversibly induces VEGF expression through both increased transcription andstabilization of the mRNA (Levy et al., 1996). Hypoxic upregulation of VEGF thus providesa compensatory mechanism by which tissues can increase their oxygenation through induc-tion of blood vessel growth. Normoxia downregulates VEGF production and leads toregression of certain newly formed blood vessels. By these opposing processes the vascu-lature becomes matched to the tissue oxygen demands (Veikkola and Alitalo, 1999).

The vascular endothelial growth factor is produced by tumor cells, macrophages, andendothelial and smooth muscle cells. It induces vascular endothelial cell migration,enhances vascular permeability, and promotes extravasation of plasma proteins from tumorvessels to form an extracellular matrix, facilitating inward migration of endothelial cells(Callagy et al., 2000). These characteristics impart selectivity to VEGF for endothelial cells.

The vascular endothelial growth factor is involved in angiogenesis in a wide variety ofbiological systems, including the female reproductive cycle, wound healing, and tissuerepair. Proliferation of blood vessels during the formation of the corpus luteum in the ovaryand during the growth of endometrial vessels in the uterus occurs upon expression of theVEGF mRNA and protein (Ferrara and Davis-Smyth, 1997). This factor is also detectedduring angiogenesis occurring at the site of embryo implantation in the uterus (Shweikiet al., 1993). In ischemic cardiac tissue, VEGF mRNA is increased, suggesting the involve-ment of this factor in the growth of collateral blood vessels (Hashimoto et al., 1994).

In addition to its role in physiological angiogenesis, VEGF is active in pathologicalneovascularization. For example, squamous cell carcinoma of the skin strongly expressesVEGF (Weninger et al., 1996). In fact, tumoral VEGF correlates with prognosis in a vari-ety of tumors, including breast cancer and malignant mesothelioma (Fig. 1.6).

Immunohistochemical Localization of Vascular Endothelial Growth Factor

The following method is recommended for immunostaining vascular endothelialgrowth factor (VEGF) (Callagy et al., 2000). Breast cancer tissues (including the invasive

Introduction 25

edge of the tumor) are fixed with formalin and embedded in paraffin. Sections thick)are mounted onto adhesive-coated slides, dried, and then deparaffinized. The sections aretreated with 3% hydrogen peroxide for 5 min to block endogenous peroxidase activity. Afterwashing in PBS, the sections are placed in 10 mM sodium citrate buffer (pH 6.0) and boiledfor 5 min in a microwave oven to unmask the antigens. They are washed in Tris buffersodium chloride (25 mM Tris-HCl [pH 7.6] and 150 nM sodium chloride) and incubated innormal goat serum (diluted 1:10 with TBS) for 30 min to block nonspecific staining.

The sections are incubated in the rabbit polyclonal anti-VEGF (Santa Biotechnology,CA) and diluted 1:100 for 30 min at room temperature. Antigen-antibody reaction isdetected using the biotin-streptavidin–based detection kit (Dako). The reaction is devel-oped using DAB+hydrogen peroxide and counterstained with Mayer’s hematoxylin.Exclusion of the primary antibody serves as a negative control. The results of this proce-dure are shown in Fig. 1.7.

Telepathology (Telemedicine)

Telepathology, introduced by Weinstein et al. (1987), is a pathology practice thatrequires telecommunication technologies to transmit digital images to distinct sites for diag-nostic, consultation, and educational purposes. Telepathology is an affordable option inplaces where a pathologist is unaffordable, such as rural hospitals that are too small to sup-port a pathologist. In addition, telepathology facilitates seeking a second opinion on a diffi-cult case. It is thought to be accurate and cost-effective, and its advantages outweigh theproblem of waiting longer to have a slide read. Also, the resolution obtainable with videomi-croscopy is thought to be adequate and appropriate for diagnosis. Telepathology is expectedto become an integral part of medical practice for practical, economic, and humane reasons.

26 Chapter 1

Telepathology can be divided into two major modalities: static imagery and dynamic(real-time) imagery. Each of these two methods has advantages and limitations. Staticimagery (the store-and-forward method) involves capturing of still images from a micro-scope and transmitting them through a point-to-point connection or by transmission con-trol protocol/internet protocol. The still digital images selected at the remote site aretransmitted at a later time for remote diagnosis. The images provided are of superior qual-ity, but the number of images is limited. It is usually not feasible to transmit the images inreal time, and the selection of images by the remote site requires two pathologists to sharethe interaction. The images can be transmitted over the Internet because bandwidth (roleof data transmission) requirements are low for the static imagery.

In static imagery, expedient delivery of high-resolution images can be achieved byattaching pathology images with an electronic mail message. This method is applicable whenreal-time consultation is not required. Despite the low cost and simplicity of static imagery,

Introduction 27

it has certain shortcomings. Because static imagery uses relatively low-resolution digitalphotomicrography, it requires high optical magnification to allow adequate examination ofdiagnostic images. This results in the need to collect and transmit multiple image files.

Furthermore, static image acquisition means that fields for imaging are preselected bya person other than the telepathology consultant, leading to unattended field selectionbiopsy error (Weinstein et al., 1997). However, high-resolution digital scanning camerasallow the acquisition of digital images up to 3,400 × 2,700 pixels of resolution. Theseimages can be captured at a relatively low optical magnification and digitally magnifiedmultiple times without visible degradation. They can be scrolled at different magnifica-tions in computer, simulating light microscopy. This high-resolution digital photomicrog-raphy and the Internet have been used for telepathological gastrointestinal biopsyconsultations (Singson et al., 1999).

In contrast to static imagery, in dynamic imagery the consultant examines a histologicalor cytological slide from a remote site by using sophisticated robotic microscopes that trans-mit real-time digital images through fast and expensive telecommunication links that providevery high band widths. According to Weinstein (1996), low-resolution dynamic images aremore useful to a pathologist than high-resolution static images. The diagnostic accuracies forstatic imaging and dynamic imaging are 88% and 96–97%, respectively. The latter range fallswithin the acceptable range for surgical pathology. Although the real-time telepathologyshows a higher diagnostic accuracy, static imagery continues to be the dominant methodused. Static imaging is adequate in those cases where tissue sampling is not a problem.

Attempts have been made to develop systems that combine the advantages of staticimagery and dynamic imagery. Such systems have been described by O’Brien et al.(1998). However, few of these systems have been implemented because of their complex-ity. Recently, a new hybrid telepathology system has been described, which achievesdynamic real-time microscopic video transmission for providing dynamic imaging. Theimplementation of this system is awaited.

Imaging standards remain an issue in telepathology. Lack of critical literature in thisfield is also a barrier to further development and acceptance of this technology. Furthermore,standards must be developed and accepted for the types of cases that will be diagnosed andfor protecting patients’ privacy. In addition, telepathology equipment is more expensive thanteleradiology equipment.

The most extensive and well-known telepathology service is part of the U.S. ArmedForces Institute of Pathology. This service offers the diagnostic evaluation of microscopic stillimages sent via e-mail (http://www.afip.org/). Recently, telemicroscopy via Internet browserssuch as Netscape Navigator and Internet Explorer was introduced by Wolf et al. (1998a, b).They reported a new concept in Internet functionality by demonstrating how Internetbrowsers with Java support can use remote control of computer-controlled devices such as anautomatic microscope. More recently, Petersen et al. (2000) reported how this technology canbe used for image transfer and communication between pathologists or research scientists.Essentially, it is based on a conventional light microscope with a video camera, which in turnis connected to a computer with a frame grabber and Internet access. This Telemic systemallows the user to show and discuss microscope images with any pathologist who is con-nected to the Internet. For inquiries about the software and information on the installation, thereader should contact the Telemic homepage at http://amba.charite.de/telemic

28 Chapter 1

FUTURE OF IMMUNOHISTOPATHOLOGY

An increasing understanding of the molecular changes associated with various tumorgroups, and the genetic variability within them, is beginning to provide important newinformation about clinical progression and prognosis (Graadt van Roggen et al., 1999).Many malignant tumors carry chromosomal aberrations detectable at a cytogenetic ormolecular level. Some of these changes are nonrandom and associated with specific tumortypes (Ladanyi, 1995). Chromosomal analysis aimed at detecting specific alterations is themost effective variable in resolving the frequent diagnostic dilemmas.

The detection and description of apparent tumor-specific genetic alterations withinthe sarcoma group are already beginning to play an increasingly important role in unrav-eling and understanding the molecular biology of tumorigenesis. For example, mutationsin the retinoblastoma gene (Rb) have been detected in a large proportion of high-grade sar-comas (Sreeckantaiah et al., 1994). Also, the coinactivation of p53 and Rb indicates thatboth genes may be involved in tumorigenesis of certain sarcomas. In fact, the identifica-tion of increasing numbers of tumor-specific genetic alterations has become an helpfuladjunct to histopathological assessment in reaching a correct diagnosis. The above obser-vations suggest that an accurate histological classification of tumor types is useful in estab-lishing meaningful clinical trials of optimal management strategies.

The relationship between immunohistopathology and surgical pathology becomesapparent when one considers that a large number of monoclonal antibodies is being pro-duced that detect cells at each stage of cancer development. These antibodies are directedagainst antigens that determine levels of proliferation, angiogenesis, proteolysis, and celladhesion (Elias, 1999). Thus, it has become possible to determine biochemical alterationsoccurring during the cell’s progression to malignancy. In addition, new oncogenes arebeing discovered at a rapid pace. These developments are helping pathologists and oncol-ogists to refine therapeutic and prognostic decisions. Furthermore, these advancements,along with increasing understanding of the molecular and cellular mechanisms of cancer,are expected to lead us to the evaluation of a person’s risk of developing cancer. The ultimategoal, of course, is to prevent cancer.

The relationship between gene expression profiles and cellular behavior in humans islargely unknown, and expression patterns of individual cell types have yet to be preciselymeasured (Emmert-Buck et al., 2000). Although we know that the human genome consistsof 32,000 genes, at present the function of only a relatively small percentage of genes isknown. However, it is hoped that our understanding of how gene expression modulates cel-lular phenotype and response to the environment will be achieved within the next fewyears or a few decades.

In June 2000, the International Human Genome Project and Celera GenomicsCorporation announced the completion of a “working draft” of the human genome sequence,the genetic code that carries the instructions allowing us to develop, grow, and live. It is pos-sible now to understand the secrets of life processes to an extraordinary degree, to personal-ize medicine and offer clues to the differences and remarkable similarities among us.

Human genome information in concert with full-length cDNA sequencing of allgenes will also lead us to an exciting new paradigm in biomedical research known asmolecular profiling (Emmert-Buck et al., 2000). Molecular profiling will facilitate theidentification of individual genes and collection of genes that mediate particular aspects of

Introduction 29

cellular physiology and pathology, thus improving our understanding and treatment ofdiseases.

I am confident that with the approach of the postgenome era, an ever-increasing num-ber of human genes will be discovered and their functions elucidated. Combined with theknowledge of human gene polymorphism, genotyping will allow prediction of the geneticpredisposition to certain diseases, such as cancer. The new millennium will usher us in anew era of disease-predictive medicine.

PREPARATION OF BUFFERS

Tris-buffered saline (TBS) (Stock solution)

Tris 303 gNaCl 450 gDistilled water 4 literAdjust pH to 7.8 185 ml HCl (32%)Distilled water to make 5 liter

Dilute 10 times with distilled water before use

Phosphate-buffered saline (PBS) (Stock solution)

Disodium hydrogen phosphate dihydrate 70.5 gPotassium dihydrogen phosphate 10.5 gNaCl 450 gDistilled water 4 literAdjust pH to 7.4Distilled water to make 5 liter

Dilute 10 times with distilled water before use

Citrate buffer for heat-induced antigen retrieval

Citric acid monohydrate 2.1 gDistilled water 4 literAdjust pH to 6.0 with 13 ml 2 N NaOH solution

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Chapter 2

Antigens and Antibodies

It is instructive to define relative terminologies. An antigen is a molecule that combines witha specific antibody but which itself may not necessarily be immunogenic. An immunogenis a cell or macromolecule that stimulates a specific immune response. An epitope (an anti-genic determinant) is the site on a complex antigenic molecule which is recognized by theantibody. An antibody (immunoglobulin) is a glycoprotein molecule produced by differen-tiated B lymphocytes when stimulated by an antigen. Immunoglobulin G (IgG) is the mostabundant class of immunoglobulins in human serum. IgG is the primary Ig molecule pro-duced during the secondary immune reaction to the antigen. Immunization is the adminis-tration of an antigen to an animal to evoke the production of antibodies. Serum is the bloodplasma from which the fibrogen has been removed. Mammalian sera contain ~8% (w/v)protein, consisting of approximately equal proportions of albumin and globulin. Antiserumis the serum containing antibodies to an antigen.

Fab is the fragment of an immunoglobulin (Ig) that binds to an antigen and is pro-duced by treating the Ig molecule with the enzyme papain. is the fragment of an Igmolecule which contains both antigen-binding sites and the disulfide bridge. It is producedby treating the Ig molecule with the enzyme. Fc fragment is the part of an Ig molecule thathas no antigen-binding activity but binds to FC receptors on phagocytes and may activatecomplement. A clone is a group of daughter cells that are produced from a single cell.Hybridoma is the cloned hybrid cells formed by the fusion of an antibody-forming cell anda malignant myeloma cell. A hybridoma grows continuously and produces antibodies of asingle specificity termed monoclonal antibodies. Affinity is the association constant at theequilibrium between an epitope and a single antigen-binding site of the antibody, inde-pendent of the number of sites. This term describes the strength and the stability of thebinding. Specificity refers to the selective binding between an antigen and its correspon-ding antibody. Titer is the measure of units of antibody per unit volume of serum. Theconcentration of the antibody is determined by titration.

ANTIGENS

An antigen is a substance that reacts specifically with receptors on the surface of lym-phocytes and with their soluble products such as antibodies. Antigens usually are large,complex protein or polysaccharide molecules with molecular weights usually greater than

31

32 Chapter 2

40,000. However, the molecular weight, for example, may vary from 15,000 (hen egg whitelysozyme) to 2,000,000 (keyhole limpet hemocyanin) daltons. Protein antigens function asthe most potent immunogens, and polysaccharide antigens rank second. For cell-mediatedimmunity, only proteins serve as immunogens. Certain nucleic acid types such as Z-DNAand other molecules can also stimulate antibody production.

Antigens can be defined on the basis of four immunological properties: immuno-genecity, antigenicity, allerogenicity, and tolerogenicity. The ability of a substance toinduce an immune response is called immunogenicity. Most antigens have a variety ofdifferent antigenic determinants (epitopes) on their surfaces, which stimulate antibodyproduction. Antigenicity is the ability of an immunogen to combine with an antibody orcell surface receptors.

Allerogenicity is the ability to induce various types of allergic responses. Allergensare immunogens that tend to activate specific types of humoral or cell-mediated responseshaving allergic manifestations (Kuby, 1992). Tolerogenicity is the capacity to induce spe-cific immunological nonresponsiveness in either the humoral or the cell-mediated systems.In other words, experimentally induced tolerance can be defined as a state in which an ani-mal fails to respond to an antigen that would normally be immunogenic. It is not knownwhether similar mechanisms generate both naturally acquired self-tolerance and experi-mentally induced tolerance.

Immunogens induce an immune response only if they are recognized as foreign (non-self). A case in point is protein bovine serum albumin, which is immunogenic in sheep butnot in cows. Most large antigens have multiple reactive sites, or epitopes, on their surfaces,which can induce production of specific antibodies. Antibodies do not recognize the wholeimmunogen but only small regions (epitopes). Each type of antibody binds to its owninducing epitope. For example, lysozyme, an enzyme that degrades the carbohydrate coatof bacteria, induces several different antibodies, each of which binds to a particular epi-tope on the lysozyme molecule (Lodish et al., 2000). Although different epitopes onlysozyme differ greatly in their chemical properties, the interaction between lysozyme andantibody is complementary in all cases. In other words, the surface of the antibody’s antigen-binding site fits into that of the corresponding epitope as if they are molded together. Theintimate contact between these two surfaces, stabilized by numerous noncovalent bonds,is responsible for the exquisite binding specificity shown by an antibody.

Epitopes

Immune cells do not interact with or recognize an entire immunogen molecule;instead, lymphocytes recognize discrete sites on the antigen called epitopes (antigenicdeterminants). Epitopes are the immunologically active regions of an immunogen whichbind to specific membrane receptors for antigen on lymphocytes or to secreted antibodies.Interaction between lymphocytes and a complex antigen may involve several levels ofantigen structure. In the case of protein antigens, the structure of an epitope may involveelements of the primary, secondary, tertiary, and even quaternary structure of the protein.In the case of polysaccharide antigens, extensive side-chain branching via glycosidicbonds affects the overall three-dimensional conformation of individual epitopes.

Epitopes are small linear sequences of amino acid residues, branched sequences of car-bohydrate, or “shape” sequences brought about by the folding of a protein molecule. The

Antigens and Antibodies 33

remaining part of the protein antigen molecule is the carrier for the epitope. An antigen suchas bovine serum albumin has several different epitopes on its surface, each of which stimu-lates the cell having the appropriate receptor. Because an epitope usually comprises asequence of approximately three to eight amino acid residues, antibodies should be regardedas site- or region-specific detection molecules instead of antigen-specific molecules.

ANTIBODIES

Immunohistochemical labeling with antibodies has become the most sensitive and pow-erful method for localizing antigens in situ and thus for characterizing cells and their com-ponents and their functions. In this context the importance of antibody specificity andselectivity for the antigen cannot be overemphasized. The specificity relies entirely on theproperties of the primary antibody, independent of the procedure used for detection. Althoughboth monoclonal and polyclonal primary antibodies can be generated or purchased, the for-mer are preferred and are in much wider use because of their far greater specificity.

The basic structure of an antibody molecule is Y-shaped, with the two tips designedto recognize and bind antigens (Fig. 2.1). The tips, through the disulfide bridge, are free tobend with respect to each other. This property increases the binding strength of the anti-body for antigens that have multiple, adjacent antigenic determinants and for antigens thatare closely packed together. The remainder of the antibody molecule enables it to interactwith other proteins, preventing undesirable company.

Antibodies are molecules secreted by terminally differentiated B cells (a type of lym-phocyte) known as plasma cells. Nearly all rabbit primary antibodies and most mousemonoclonal antibodies are immunoglobulins (Igs). There are five classes of Igs that differstructurally and functionally. Immunoglobulin G (IgG) molecules are the major class ofIgs in the blood, which are predominantly produced in the secondary immune response.

Monoclonal antibodies have been termed magic bullets and hailed in publications as thecure for cancer. Belief in this idea was strengthened by the successful clinical results of mouse

34 Chapter 2

anti-idiotypic monoclonal antibodies in the treatment of lymphomas and leukemias (Levyand Miller, 1983) and by FDA approval in 1986 of the OKT3 anti-CD3 mouse monoclonalantibody for acute renal transplant rejection (Shield et al., 1996). However, this excessiveoptimism has been questioned because of adverse clinical and laboratory findings. For exam-ple, when rodent monoclonal antibodies were used therapeutically, a human antimurine anti-body response developed in up to 50% of treated patients (Khazaeli et al., 1994).

Effector functions of mouse antibodies also have proven less efficient in the humancontext (Gavilondo and Larrick, 2000). The biological half-life of these antibodies isshorter than that of human immunoglobulins. This characteristic of mouse antibodies lim-its their usefulness. These limitations can theoretically be overcome by using monoclonalantibodies of human origin (Thompson, 1988); however, human monoclonal antibodiesfrom hybridomas and lymphocyte cell lines are very difficult to generate. Nevertheless,beginning in 1994, the FDA approved a number of antibodies to combat human diseases,including follicular non-Hodgkin’s B cell lymphoma, breast cancer, and rheumatic arthri-tis (Grillo-Lopez et al., 1999; Weiner, 1999; Maini et al., 1999).

Polyclonal Antibodies

Both polyclonal and monoclonal antibodies have advantages and limitations withregard to their generation, specificity, cost, and overall applications. Polyclonal antibodiespossess higher affinity and wider reactivity but lower specificity. They have the advantageof detecting many types of epitopes and recognizing antigens of different orientations.Polyclonal antibodies show greater stability at varying pH levels and salt concentrationsand are more useful for preadsorption controls. They are simpler to produce in a shorterduration, and there is no risk of loss of clones. In addition, large animals (e.g., rabbits andhorses) can be used to recover large volumes of antibody-rich serum. However, a freshbatch of the serum is required when the original stock is exhausted. This replacementresults in batch-to-batch variation, which may result in differences in antibody reactivityand titer (Nelson et al., 2000). Such differences result in a lack of reproducibility.

Polyclonal antibodies are composed of multiple species of immunoglobulins directedtoward several epitopes within a particular antigenic molecule. Moreover, only a minorproportion of the antibody present in the polyclonal antiserum is specific for the immu-nizing antigen. The remainder may consist either of antibodies produced by the animal inthe past in response to previous antigenic stimuli or of antibodies against contaminatingantigens present in the immunizing preparation (Mason et al., 1983). Even the antibodiesin the polyclonal antiserum that are specific for the immunizing antigen are usually het-erogeneous and are directed against a number of different epitopes on the immunizing anti-gen.

However, although whole sera or whole IgG fractions of polyclonal antibodies oftenhave problems, affinity purification against an antigen affinity column can dramaticallyimprove the usefulness of the polyclonal reagents. Thus, a mixture of isoforms of antibod-ies to different epitopes is obtained. These epitopes are still relatively unique to the antigeninvolved. The main advantage is that 100% of the antibody in these preparations reacts withthe antigen, often at multiple and therefore additive sites. This approach is analogous tomixing monoclonal antibodies to label different epitopes together. Although such reagents

Antigens and Antibodies 35

are not commonly commercially available, their usefulness in immunohistochemistryshould not be minimized.

The procedure to produce such reagents requires the ability to prepare significantamounts of the pure antigen, either a purified protein (or carbohydrate) or even a complexpeptide. The antigen is covalently linked to the beads of a column (usually cyanogen bro-mide–activated Sepharose). The antibody preparation is passed over the column and theantibody reactive with the antigen sticks. The excess (usually 99% of the total IgG) (flowthrough) is then washed away from the column, and the specific antibody is eluted fromthe column selectively using acid, base, thiocyanate, or a high salt such as magnesiumchloride.

The eluted antibody is then neutralized and dialyzed to remove the salts. Such anti-body usually represents only ~1% of the total IgG in the serum. This procedure is differ-ent from the so-called affinity-purified antibody in which protein A or protein G is used;the latter purifies IgG only and has no meaning in relation to selective antigen reactivity.

Production of Polyclonal Antiserum

The following procedure can be used to produce polyclonal antiserum in rabbits (Beltzand Burd, 1989). Preimmune blood is removed from the rabbit’s ear vein for later use in con-trol experiments. The rabbit is immunized with of the antigen. If a carrier pro-tein is used, the carrier and the antigen are injected together. One milliliter of the antigen(including the carrier) in buffer and 1 ml of Freund’s complete adjuvant are emulsified com-pletely and injected subcutaneously into several locations on the rabbit’s back. Freund’scomplete adjuvant contains ingredients that increase the rabbit’s immune response. Thecomplete adjuvant includes saline, emulsifying agent, mineral oil, and killed mycobacteria.

The mixture is injected once a week for 3 weeks, and then the animal is maintainedfor 3 weeks without additional injections. Approximately 25–40 ml of blood is removedfrom the animal’s ear vein to test for antibodies. One week after the first bleed the rabbitis boosted with half the antigen amount used earlier along with incomplete adjuvant;incomplete adjuvant does not contain the mycobacteria. Serum is again removed 2 weeksafter this injection and tested for antibody response. The enzyme-linked immunosorbentassay is used to determine if the titer (or antibody concentration) of the serum is suffi-ciently high to establish antibody binding to the antigen. This 3-week cycle is repeated aslong as necessary to obtain the antibodies desired.

The peak response is generally achieved at the sixth to eighth injection (~5–7months) after the initial immunization. If the rabbit is to be sacrificed, the final bleedingcan yield 50–70 ml of serum. When the animal has made antibody in sufficient quantity,its serum can be used directly as the immunohistochemical reagent. It can be purified usingan affinity column to remove nonimmunoglobulin proteins, extraneous antibodies, or toselect antibodies that recognize a specific antigen. The use of affinity purified antibodiesreduces nonspecific, background staining.

Affinity Chromatography

Antibody affinity chromatography is employed to isolate antigen-specific antibodies.The most common affinity matrix for coupling of molecules is cyanogen bromide–activated

36 Chapter 2

Sepharose. The following procedure can be used to purify antibodies raised against aparticular protein (Javois, 1999).

1.

2.

3.

4.

5.

6.7.

8.

9.

Sprinkle 1 g of cyanogen bromide–activated Sepharose 4B (Pharmacia-LKB,Piscataway, NJ) over 20 ml of 10 mM HC1. The gel swells immediately. Onegram of dry gel yields ~3.5 ml of hydrated matrix.Wash the gel on a 50-ml coarse, sintered glass funnel four times with 50 ml of10 mM HCl by repeatedly suspending the matrix in the HCl solution, and thendrain using vacuum suction. These washing steps remove the additives present inthe dry matrix.Suspend 5 mg of rat immunoglobulin in 5 ml of coupling buffer A (100 mM

500 mM NaCl, and 200 mM glycine, pH 8.0).Add this supension to the gel and mix by inversion overnight at 4°C in a capped15-ml polycarbonate tube. Avoid mechanically stirring the gel to avoid damagingthe gel matrix.Pour the matrix into a sintered glass funnel and drain the gel; save the eluate toestimate the amount of antibody couple. Estimate the amount of protein bound tothe column by subtracting the quantity of IgG that is eluted. The eluate shouldnot contain more than 20% of the applied protein concentration.Wash the matrix with 100 ml of coupling buffer A to remove any unbound ligand.Suspend the matrix overnight at 4°C in 45 ml of 200 mM glycine (pH 8.0) andmix by inversion in a 50-ml capped tube to block any unreacted groups.Drain and wash the gel with three cycles of alternating pH. First, suspend thedrained gel in 50 ml of 100 mM sodium acetate (pH 4.0) and 500 mM of NaCl.Drain with vacuum suction and wash with 50 ml of coupling buffer A. Drain andrepeat the alternating pH washes twice.Suspend the gel in 20 ml of BBS buffer (dissolve 247.3 g of boric acid, 187 g ofNaCl, and 75 ml of 10 M NaOH in 4 liters of distilled water, pH 8.0). The matrixis then ready for use in column chromatography.

10.

11.

12.13.

14.

15.

16.17.18.

Pack the matrix in a Poly Prep column (Bio-Rad), which can be stored at 4°C; itshould not be allowed to warm up or dry out.Attach the column outlet to a peristaltic pump, and wash the column with 5 mlof BBS at 0.5 ml/min.Drain most of the BBS, leaving ~0.5 ml on top of the gel bed.Apply 15 ml of rabbit antirat IgG to the column, and circulate the solutionthrough the matrix at 0.2 ml/min for 3 hr at 4°C.Drain the column as in step 12 and save the eluate, which may still contain someof the desired antibodies. The titer of the eluate can be tested for reapplication tothe gel at the end of the first purification, although this may not be necessary.Wash the matrix with ~10 column volumes of BBS until the absorbance of theeluate is <0.02 at 280 nm compared to the column buffer.Remove the bound antibody with 5 ml of 100 mM glycine (pH 3.0) at 0.5 ml/min.Collect 1-ml fractions into tubes containing of 1 M Tris-HCl buffer (pH 8.0).Pool the IgG-containing samples and concentrate as necessary. The samples con-taining the highest absorbance at 280 nm should be pooled. Any precipitated anti-bodies can be removed by centrifugation at 10,000 g for 30 min at 4°C. The IgGs

Antigens and Antibodies 37

can be concentrated and stored at 4°C for weeks or at – 80°C for months andyears. Avoid repeated freezing and thawing, which may denature the proteins.

Monoclonal Antibodies

Unlike polyclonal antibodies, monoclonal antibodies are directed against single epitopesconsisting of very short sequences of amino acids. These antibodies are able to provide invalu-able information about the molecular conformation of a particular epitope within a given anti-gen. However, even though a monoclonal antibody can recognize multiple molecules, itsspecificity remains intact. This phenomenon is due to the presence of very similar epitopes indifferent peptides and proteins. This subject is discussed in detail later in this chapter.

The development of monoclonal antibodies has led to a new era of enhanced diagnos-tic and therapeutic modalities. The impact of this development on diagnostic imaging tech-nologies has been dramatic, resulting in a revolution in modern diagnostic medicine. Recentadvances in recombinant antigen preparation have further advanced the usefulness of thesemethods; almost any protein can be engineered to be expressed in nonnatural host cells.

If monoclonal antibodies to a specific region or a specific epitope of an antigen aredesired and the amino acid sequence of this region is known, synthetic peptides can be pre-pared for animal injection. In fact, synthetic peptides are currently being used for the injec-tion of animals to generate antibodies for a specific region or a specific region epitope ofan antigen. As a result, monoclonal antibodies of high affinity and specificity are beingproduced at a fast pace. These antibodies are being effectively employed for the detectionand analysis of antigens, and are playing a key role in clinical diagnostic medicine. Thesedevelopments are partially responsible for the exponential growth in our understanding ofthe physiology of human diseases.

Monoclonal antibodies are produced only when necessary because their generation isdifficult, time-consuming, and frustrating. Nevertheless, the most important reason for pre-ferring them is their exquisite specificity, as ideally they recognize only one type of epi-tope. Moreover, these antibodies show a high biological half-life in blood and othertissues, rendering them effective for prophylactic use. The toxicity of infused monoclonalantibodies is expected to be low because of their biological nature. Thus, these antibodiesare being used extensively and successfully in routine pathology laboratories to aid in theclinical diagnosis and treatment of malignant diseases.

To produce monoclonal antibodies, lower doses of antigens are required forimmunoresponse. The continuous culture of B cell hybridomas yields a reproducible andpotentially inexhaustible supply of the monoclonal antibody; all batches are homogenous.Consequently, these antibodies allow the development of standardized procedures forclinical diagnosis. In addition, monoclonal antibodies are ideal for a complex mixture ofantigens (e.g., membrane antigens), for scarce antigens, or when attempting to detectunique epitopes where purification is difficult or impossible (Beltz and Burd, 1989).Monoclonal antibodies do not have high affinities and so generally must be used at lowerdilutions. Moreover, monoclonal antibodies are more expensive to generate or purchasethan polyclonal antibodies. Exceptions to some of the advantages and limitations ofpolyclonal and monoclonal antibodies listed above are not uncommon. A large number ofmonoclonal antibodies are commercially available (see page 50).

38 Chapter 2

Specificity of Monoclonal Antibodies

Affinity is an important feature of monoclonal antibodies and is especially importantfor those antibodies used in clinical diagnosis. The measurement of antibody affinity pro-vides an indication as to the strength with which a monoclonal antibody specifically bindsto its target molecule. Another important characteristic of a monoclonal antibody is thespecific location or region on the antigen to which the antibody binds. Although mostmonoclonal antibodies exhibit strong affinity and high specificity for epitopes, this is nottrue for all. In this context, the mechanisms responsible for reaction of antibodies withantigens in the tissues that have been processed for immunohistochemistry are exceedinglycomplex. The processing conditions under which an antigen is exposed to an antibody arecritical in determining the specificity of an antibody.

The processing conditions employed determine the reactive epitopes. Under certainconditions the epitope recognized may be other than that under study. Various aspects ofantibody–antigen interaction, including the role of fixation, are discussed below.Monoclonal antibodies are directed against epitopes consisting of a small number of aminoacids, which can be part of several types of proteins and peptides. Therefore, it is notuncommon for some monoclonal antibodies to label unrelated antigens in different tissues.Several examples are cited below. A monoclonal antibody against a monocyte-macrophageprotein also binds to enamel proteins (Nakamura et al., 1991). Several monoclonal andpolyclonal antibodies against osteocalcin (a bone protein) also cross-react with epitopes oncultured skin fibroblasts (Bradbeer et al., 1994). An anti–human proinsulin antibody cross-reacts with both insulin and glucagon-secreting cells (Bendayan, 1995). Another exampleis the monoclonal antibody (C219) against the multidrug transporter (P-glycoprotein),which also reacts with the slow-twitch skeletal muscle myosin (Thiebaut et al., 1989).

Because most of the immunohistological studies, including those cited above, arecarried out using chemically fixed tissues, it is difficult to be certain whether the immuno-reactivity is owing to shared epitopes (cross-reactivity) or epitopes resulting from proteincrosslinking during fixation with an aldehyde. A recent study has demonstrated that antivi-mentin antibody (V9-S) cross-reacts with amelogenins in the glutaraldehyde-fixed rathemimandibles (Josephsen et al., 1999). This immunoreaction is thought to be directedagainst the epitope generated by crosslinking of enamel proteins during fixation becausethis antibody binding occurs only after fixation. Moreover, any significant homologybetween vimentin and amelogenin is absent. Another example is PC10 antibody, whichreacts with PCNA only after fixation (Willingham, 1999). Such reactions have been callednonspecific but selective. However, as long as these reactions are useful, they are both spe-cific and selective.

Considering the possibility of a monoclonal antibody reacting with more than onetype of antigen or epitope, the positive staining should be interpreted carefully.Nonspecificity in the immunostaining can render correct interpretation difficult. This prob-lem can arise due to nonspecific binding of antibodies and reagents, cross-reacting endoge-nous antibodies, and the presence of the same or similar epitopes in different antigens. Inother words, when monoclonal antibodies show unexpected cross-reactivity, it is not clearwhether this phenomenon is due to the presence of the same molecule in two different cellpopulations or to the presence of identical (or very similar) epitopes on quite different mol-ecules. These difficulties are encountered in both frozen and paraffin sections. It should be

Antigens and Antibodies 39

noted, however, that the major effect of aldehyde fixation on the reaction of the antibodywith the antigen is the inaccessibility of the antigen to the antibody. In other words, thecrosslinking of cytosol proteins introduced by aldehyde fixatives creates a barrier for theantibody molecule to penetrate the cell and react with the antigen (epitope).

Monoclonal antibodies generated against epitopes react with the native conformationof the antigen, which may be preserved by fixation. These antibodies are useful forimmunohistochemistry, provided antigens are accessible to antibodies. In contrast, someother monoclonal antibodies preferentially react with denatured antigens after treatmentwith denaturing agents such as sodium dodecyl sulfate (SDS). Such antibodies generatedby immunizations with isolated peptides usually are not well suited for immunohisto-chemistry (Willingham, 1999). Based on these observations, it is likely that most mono-clonal antibodies are reactive under certain appropriate conditions used to prepare andincubate the specimens of interest. There are still other antibodies that are reactive withboth native and denatured conformations of proteins. It is thought that these antibodies rec-ognize peptide sequences exposed when proteins are denatured. On the other hand, poly-clonal antibodies do not require optimal processing condition because a polyclonalantibody recognizes multiple epitopes. These antibodies may recognize an epitope otherthan that under study.

MIB-1 Monoclonal Antibody

Because MIB-1 monoclonal antibody is used extensively to determine the cell prolif-eration index, its applications are discussed below. This antibody detects the nuclear anti-gen Ki-67 expressed in proliferating cells but not in resting cells. The antibody reacts withthe nuclei of cells in (first gap), S (DNA synthesis), (second gap), and M (mito-sis) phases, but not in the or quiescent phases. The use of MIB-1 antibody is one of thesimplest and most reliable labeling techniques for assessing the rate of proliferation of aneoplastic cell population. Thus, the antibody can be used to assess the growth fraction(i.e., the number of cells in cell cycle) of normal, reactive, and neoplastic tissues.

However, be aware that in spite of the usefulness of the MIB-1 antibody in assessingthe rate of cell proliferation, the classification of cancers (e.g., breast cancer) by the sizeof the primary tumor and the presence and extent of lymph node metastases does not ade-quately explain differences in the clinical outcome of individual patients. Cell proliferationindices are commonly used, along with other diagnostic parameters, to estimate the risk ofrecurrence of a cancer for individual patients. Therefore, it is important to understand therelationship between various indices of proliferation such as MIB-1 labeling index anddetection by either in situ hybridization or polymerase chain reaction. This approach willlead to quality assurance in diagnosis.

Considerable evidence is available indicating a close correlation between the growthfraction of proliferating cells, as measured by other techniques, and the number of cellsstained using MIB-1 antibody. Thus, MIB-1 labeling index can be used in conjunction withother histological features to determine the potentially aggressive behavior of a tumor. Forexample, MIB-1 labeling index of the growth fraction correlates significantly with thebromodeoxyuridine (BrdU) index of DNA synthesis in S phase, and both indices correlatewell with other parameters of tumor aggressiveness (Goodson et al., 1998). In many cases,

40 Chapter 2

but not in all, a correlation also exists between the label index and patient survival.However, although these indices are related, clinical comparison is necessary to determinewhich is the better prognostic marker for human breast cancer and other humancancers.

A brief comment on the usefulness of the BrdU method is relevant. Currently, immuno-histochemical detection of exogenously injected 5-bromodeoxyuridine several hours prior toanimal sacrifice is widely used to assess proliferation state in murine tissues. This nucleotide-analog probe is integrated into the DNA of replicating cells during S phase (Selden et al.,1993). The probe can be subsequently detected immunohistochemically in paraffin-embeddedtissue sections. However, the proportion of anti-BrdU stained cells is variable depending onthe duration and frequency of BrdU injections before sacrificing the animal.

A number of examples given below support the prognostic value of MIB-1 antibody.Evaluation of proliferation index in malignant mesothelioma can be performed using theMIB-1 antibody (Comin et al., 2000). In this case a correlation is found between the label-ing index and survival. Malignant mesothelioma is a rare, aggressive, and frequently lethaltumor, usually associated with asbestos exposure. It should be noted that numerous prog-nostic factors (e.g., age, tumor stage, asbestos exposure, performance status, histologicalsubtype, tumor angiogenesis, and proliferation index) are correlated with survival. Anotherexample is the high proliferative index found in the esophageal small cell carcinoma usingMIB-1 antibody, indicating aggressive behavior (Lam et al., 2000).

Also, Ki-67 antigen labeling with MIB-1 antibody is a reliable method for estimatingthe proliferative activity in uveal melanomas after proton beam irradiation (Chiquet et al.,2000). The Ki-67 score is significantly correlated with prognostic variables (mitotic indexand histological largest tumor diameter) and with radiation effects after proton beam irra-diation. Furthermore, a higher Ki-67 score is found in uveal melanomas with metastasisthan in tumors without metastatic evolution. Uveal melanoma is the most common primaryadult ocular malignancy. Their ability to metastasize is well recognized. It should be notedthat these views have not been accepted universally.

Another example is the labeling of Ki-67 with MIB-1 antibody in benign and malig-nant apocrine lesions of the breast, which facilitates differentiation between benign andmalignant breast apocrine lesions (Moriya et al., 2000). Both the number of positive casesand the percentage of positive tumor cells are significantly higher in malignant cases thanin benign apocrine cases. Thus, Ki-67 immunohistochemistry can be an auxiliary methodfor determining the possible biological behavior of lesions and for the diagnosis and prog-nosis of patients with apocrine lesions of the breast.

MIB-1 immunostaining in conjunction with microwave antigen retrieval is a benefi-cial adjunctive test when the morphological features are suggestive but not diagnostic forvulvar condyloma acuminatum (Pirog et al., 2000). Histopathological confirmation ofcondyloma acuminatum implies human papillomavirus (HPV) infection, which is sexuallytransmitted and confers an increased risk for synchronous or subsequent HPV-associatedwartlike lesions elsewhere in the female genital tract. However, histopathological diagno-sis of condyloma acuminatum is often based on architectural features that are not specificfor HPV infection.

To avoid this problem, the application of MIB-1 antibody is useful in the differentialdiagnosis of benign exophytic vulvar lesions because HPV-associated lesions show

Antigens and Antibodies 41

increased cellular proliferation. Limiting the diagnosis of condyloma to only those lesionswith koilocytotic atypia will result in underdiagnosis, and categorizing equivocal wartlikelesions of the vulva as c/w condyloma acuminatum is associated with substantial over-diagnosis. Complete concordance was found between MIB-1 positivity and detection ofHPV by both in situ hybridization and polymerase chain reaction (Pirog et al., 2000);however, MIB-1 positivity is more sensitive than the other two techniques.

Antibody MIB-1 also shows cross-reactivity. It has been demonstrated, for example,that this antibody shows strong staining of cell membrane and cytoplasm in hyalinizingtrabecular adenoma of the thyroid gland (Hirokawa and Carney, 2000). In contrast, thisantibody does not show similar immunoreactivity with papillary carcinoma. This informa-tion provides a morphological difference between hyalinizing trabecular adenoma andpapillary carcinoma, which is important because it has been suggested that these two typesof tissues are closely related tumors (Fonseca et al., 1997). This benign tumor does sharehistological features, such as intranuclear cytoplasmic invaginations, nuclear grooves, andpsammoma body–like formations, with papillary carcinoma. Thus, MIB-1 antibody can bediagnostically useful in differentiating the benign tumor from papillary carcinoma.

Note that MIB-1 antibody fails to react with Ki-67 antigen in the tissue fixed withformaldehyde or Kryofix in the absence of heat pretreatment. Because Kryofix is a non-protein, crosslinking fixative, breakdown of protein crosslinkages is not responsible for theavailability of Ki-67 antigen in this case. On the other hand, MIB-1 antibody readily reactswith Ki-67 antigen in tissue fixed with either of these two fixatives with heat pretreatment.It is apparent that in this case the effect of heat treatment is due to factors other than break-down of protein crosslinkages.

Although MIB-1 antibody is a reliable tool for determining proliferating cells inhuman tissues, it does not react with the homologous mouse antigen. Therefore, this anti-body is useless in experimental pathology using mice as the model system. Because theuse of murine tumor models has steadily increased, there is a growing need for a prolifer-ation marker for routinely processed paraffin-embedded murine tissues. Such a marker ismonoclonal antibody MIB-5, which is raised against bacterially expressed parts of thehuman Ki-67 cDNA (Schlüter et al., 1993; Gerlach et al., 1997). Recently, Birner et al.(2001) have shown that MIB-5 detects Ki-67 antigen in formalin-fixed, paraffin-embeddedmurine tissues, equivalent to MIB-1 staining of human tissues.

Production of Monoclonal Antibodies

Before presenting the procedure for generating monoclonal antibodies, it is relevantto define the terms hybridoma and monoclonal antibody. When an immune response isprovoked by an immunogen, numerous antibodies are produced against different parts orregions of the immunogen. These are termed antigenic determinants, or epitopes, and theyusually consist of six to eight amino acids. Most antibodies recognize and interact with athree-dimensional shape of an epitope composed of discontinuous residues broughttogether by the folding of a molecule (usually a protein antigen). Antibodies can also rec-ognize linear stretches of amino acids or continuous residues (an epitope). An antibody ofunique specificity derived from a single B cell is termed a monoclonal antibody.

42 Chapter 2

An immunogen induces antibodies from many B cell clones, producing a polyclonalantibody response. In contrast, the propagation of an isolated B cell clone produces anantibody of single specificity. However, the problem is that in tissue culture medium,B cells die within a few days of their isolation from, for example, a mouse spleen. Tocircumvent this problem, immortality can be conferred on B cells by means of viral trans-formation; Epstein-Barr virus can be used. Alternatively, fusion to cancerous cells is car-ried out to generate hybrids or hybridomas. Generally, the former procedure is used toimmortalize peripheral blood B cells and produce human monoclonal antibodies, whilemyeloma cells are used to produce murine monoclonal antibodies.

Although several recombinant procedures can be employed to produce monoclonalantibodies, the following protocol is used to generate murine monoclonal antibodies. Itconsists of four steps: immunization, fusion and selection, screening, and characterization(Nelson et al., 2000).

1. Immunization. Immunogen protein linked to a carrier protein (e.g.,keyhole limpet hemocyanin) is used for the primary immunization of Balb/c mice. Animmunogen is delivered in conjunction with Freund’s complete adjuvant. Adjuvants areused to enhance the stimulation of antibody production. They act by making the antigeneither more particulate or more insoluble, thus holding the antigen in a depot and releas-ing it slowly over a long period of time. These substances may also stimulate the prolifer-ation of B cell precursors in spleen, lymph nodes, and liver. Freund’s medium is a mixtureof mineral oil and antigen that is emulsified in lanolin and to which killed tubercle bacillihave been added. The latter addition increases the efficiency of the adjuvant by stimulat-ing the proliferation of macrophages. Freund’s adjuvant is unsuitable for human use.

Two milliliters of the adjuvant is placed into a 13 × 100 mm test tube. A small quan-tity of antigen is added and emulsified with a 5-ml syringe and a 20-gauge needle. Smallamounts of antigen continue to be added. Small amounts of antigen are added and emul-sification is continued until a total of 2 ml of antigen has been added. The emulsion mustbe water-in-oil (Burrell and Lewis, 1987); it is satisfactory when a drop placed on a watersurface does not spread. For rabbits, 2 ml of this preparation is injected intramuscularly ineach hind leg or subcutaneously in each of two sites at the back of the neck. Animals startresponding after 3 to 4 weeks. Regular boosting may be needed to augment polyclonalresponse, which is monitored using tail bleeds that provide sufficient serum to make surethe antibody titer to a desired antigen, using the enzyme-linked immunosorbent assay(ELISA) detailed below.

Ten wells in a row of a plastic microtiter plate are each coated with of oval-bumin diluted coating buffer:

Coating buffer

Distilled waterAdjust pH to

3.18g5.84 g1,000 ml9.5

Well 11 is skipped and well 12 is coated with of the antigen. Wells in the secondrow are filled with of coating buffer to serve as blanks. Incubation is carried out for30 min at 37°C.

Antigens and Antibodies 43

While the incubation is continuing, an antiovalbumin is titrated beginning at 1/50through 10 dilutions in 0.25-ml quantities. The serum is titrated in the diluting buffer witha micropipette.

Diluting buffer

Tween 20NaClDistilled waterAdjust pH to

0.5 ml8.5 g1,000 ml7.2

To raise pH, add 30 ml of to lower pH, add 100 ml of this solution. Allwells in a row are carefully rinsed with a wash buffer simultaneously using a well-washingdevice (Nunc Immunowash).

Wash buffer is the same as diluting buffer but without Tween 20.The rinsing is done at least eight times, and all buffer after the last rinse is removed.

Starting with the last serum dilution, is transferred to well 10 of each row. Then,of the next serum dilution is transferred to well 9 of each row, and so on until one

has reached the starting dilution, of which is transferred to each well 1. Incubationis carried out for 30 min at 37°C. This is followed by thorough rinsing with the wash bufferusing a well-washing device as indicated above.

One hundred microliters of commercial goat antirabbit globulin conjugated to horse-radish peroxidase (diluted 1/500 diluting buffer) is added to every well. Incubation is donefor 30 min at 37°C, followed by washing, and of peroxidase substrate in freshlyprepared substrate buffer is added to each well.

Substrate buffer

Citric acidDistilled waterAdjust pH to

11.85g11.73 g1,000 ml4.0

Substrate

Ten milligrams of 2,2´-azino-di-(3-ethylbenzthiazoline sulfonic acid) diammoniumsalt is dissolved in 50 ml of substrate buffer containing of 30% hydrogen peroxide.Only freshly prepared substrate should be used. Incubation is carried out for 30 min at 37°C,followed by visual plate reading on a 1+ to 4+ basis or at if a microtiter platereader is available. The optical density of the instrument is set to zero with the antigen con-trol (well 12 in the first row). Any optical density in the antibody controls (bottom row) issubtracted from the corresponding test well above.

The proteins are delivered subcutaneously. Regular boosting is needed to augmentpolyclonal response, which is monitored using tail bleeds that provide sufficient serum tomake sure the antibody titer to a desired antigen, using the enzyme-linked immunosorbentassay (ELISA). Boosting also encourages immunoglobulin class switching and the gener-ation of higher affinity antibodies through somatic hypermutation. Generally, IgG mono-clonal antibodies are preferred because they are less prone to degradation and more useful

44 Chapter 2

as therapeutic reagents. Antigenically responding B cells are removed aseptically from thespleen or lymph node to obtain cells for hybridization.

2. Fusion and selection. The murine splenic B cells are fused with histocompatiblemyeloma cells such as Sp2/0. The myeloma cells are preselected for a deficiency in theenzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT) by culturing inmedium containing 8-azaguanine. The B cells are mixed with HGPRT-negative myelomacells and a fusing agent such as polyethylene glycol. The mixing and centrifugation stepsgenerate myeloma-splenic B cell hybridomas. These hybrid cells are plated into tissue cul-ture wells. Unfused myeloma cells are removed using a selective medium containinghypoxanthine, aminopterin, and thymidine (HAT); all unfused myeloma cells will die.Hybridomas are carefully examined with an inverted microscope. Once established, thehybridoma colony will continue to grow in the culture medium (such as RPMI-1640 con-taining antibiotics and fetal bovine serum) and produce antibodies. Approximately 1 monthpost fusion, hybridomas can be propagated in HT medium (hypoxanthine and thymidine).

3. Screening. Primary screening is necessary to eliminate nonspecific hybridomas assoon as possible. Screening is also used to test the hybridoma culture supernatant for anti-body reactivity and specificity. As an example, an Epstein-Barr virus associated protein iscoated onto plastic ELISA plates. After incubation of hybridoma culture supernatant, sec-ondary enzyme-labeled conjugate and chromogenic substrate, a colored product indicatesa positive hybridoma. Alternatively, immunocytochemical screening can be used. It ispreferable to test hybridomas when at least three-quarters of them are confluent.

4. Characterization. The reactivity, specificity, and cross-reactivity of the potentialmonoclonal antibody can be analyzed by using culture supernatant or a purified immu-noglobulin preparation. It may be necessary to redone hybridomas by limiting dilutionbecause the original colony might contain at least two populations of fused B cells. In theabsence of such an analysis, the presence of antibodies of different class, specificity, andaffinity might yield ambiguous results. Characterization also provides the opportunity totest against a wide panel of related antigens or tissue preparations. This is important espe-cially for histopathological studies. Once the purification of the hybridoma is established,bulk production of a monoclonal antibody can be obtained using surface-expanded tissueculture flasks. It should be noted, however, that although a hybridoma may be the fusedproduct of a single B cell and produce a monoclonal antibody of refined specificity, suchan antibody in some cases can cross-react with other antigens or exhibit dual or multiplespecificity. The phenomenon of cross-reactivity is discussed on page 48.

It is relevant to explain the use of the letters CD as a prefix to monoclonal antibodies.Lymphocytes possess many different surface proteins, each of which possesses many dis-tinct epitopes. To classify these lymphocyte antigens, a numbering system has been estab-lished that clusters molecules having similar epitopes. Thus, all monoclonal antibodies thatdetect the epitopes on a single antigen are assigned to a numbered cluster of differentia-tion (CD). In most cases a defined CD denotes a protein of specific function. For example,the protein called CD4 is associated with cells (lymphocytes) that help the immuneresponse, while CD8 is found on cells that suppress the immune response.

Bivalent and Bispecific Monoclonal Antibodies in Cancer Therapy

Bivalent and bispecific monoclonal antibodies have many practical applications,including immunodiagnosis and immunotherapy. Bivalency can allow antibodies to bind

Antigens and Antibodies 45

to multimeric antigen with strong avidity, and bispecificity facilitates the crosslinking oftwo antigens, for example, in recruiting cytotoxic T cells to mediate killing of a tumor cell.Bivalent (IgG) antibodies can be derived from hybridomas, and bispecific antibodies byfusion of two hybridomas with two different specificities. Bispecific antibodies can formuseful immunotherapeutic tools in cancer treatment. The limited space available in thisvolume does not allow detailed discussion of the applications of these antibodies.Important in vivo animal studies, which administer bispecific antibodies, and clinical tri-als using these antibodies are listed by Koelemij et al. (1999).

Bispecific antibodies have been known for a long time. The first antibody with dualspecificity was described approximately 40 years ago (Nisonoff and Rivers, 1961). Thepotential usefulness of these antibodies in the treatment of malignancies becomes clearwhen one considers that some therapeutic applications of monoclonal antibodies inpatients with cancer have shown disappointing results. However, in general, monoclonalantibodies are thought to achieve antitumor effects by inducing antibody-dependent cellu-lar toxicity or complement-mediated cytotoxicity. Considerable experience has beengained in using monoclonal antibodies in patients with cancer, especially in the treatmentof hematological malignancies (Matthews et al., 1995). Nevertheless, the application ofmany monoclonal antibodies does not completely eliminate tumor cells. The cell surfaceexpression of complement-deactivating molecules is thought to be the escape mechanismused by tumor cells in this incomplete elimination.

Because many potentially useful monoclonal antibodies do not possess the appropri-ate isotype and so are unable to activate human complement and/or trigger on humancells, treatment strategies are needed. One such strategy is the application of bispecificmonoclonal antibodies that exploit the specificity of monoclonal antibody and ensureactivation of cellular cytotoxic mechanisms (Fanger et al., 1993).

Bispecific monoclonal antibodies are artificially developed antibodies with antigen-binding sites physically linked to different specificities. It is thought that bispecific mono-clonal antibodies activate the cellular immune response by crosslinking immune cells totumor cells, thus circumventing the proper structures for tumor cell–immune cell interac-tions (Koelemij et al., 1999). These antibodies are effective in low concentrations in vivo.For example, Kufer et al. (1996) have combined the anti-CD3 specificity directed againstT cells in a bispecific monoclonal antibody, with the specificity against the tumor-associated17-1A antigen. This antibody could be a major improvement, for example, in the therapyfor disseminated micrometastatic tumor cells.

Bispecific monoclonal antibodies are being evaluated in phase I and II studies in avariety of malignant diseases in the fields of hematooncology and solid tumors. It is likelythat in the next decade immunotherapy using bispecific monoclonal antibodies will have aplace, complementary to the current modalities such as surgery, chemotherapy, hormonetherapy, and radiation, in the treatment of malignancies.

Development of Bispecific Antibodies

The earliest bispecific antibodies were obtained by dimerization after mild oxidationof a mixture of Fab fragments of polyclonal antibodies of two different specificities.Bispecific antibodies were also obtained by crosslinking two monoclonal antibodies usingsuccinimidyl-3-2-pyridyldithiol-propionate, resulting in heteroaggregates of two mono-clonal antibodies (Staerz et al., 1985). Although this is an easy method with a high yield,

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the products are heterogeneous, ill defined, and large. Hybrid antibodies were also pro-duced by chemical processing of Fab fragments to reconstitute (hybrid) dimers (Brennanet al., 1985). These antibodies are free of mono specific contaminants and thus avoid manyunwanted side reactions.

The most commonly used technique to produce bispecific antibodies from twomonoclonal antibodies is by fusing two hybridoma cell lines by conventional cell fusionprocedure (Staerz and Bevan, 1986). These cells produce all possible combinations of theheavy and light chains of both antibodies, including the desired bispecific antibody. Alimitation is that only part of the antibodies is the desired bispecific monoclonal antibody;therefore, further purification is necessary (Van Ravenswaay et al., 1993).

Currently, mostly molecular biological techniques are used to obtain bispecific mono-clonal antibodies. By focusing on a genetic approach, molecules having the appropriatebinding regions as well as bispecificity can be prepared. For example, a so-called leucinezipper technique has been employed for producing bispecific monoclonal antibodies(Kostelny et al, 1992). Leucine zipper peptides of the transcription factors Fos and Junpreferentially form heterodimers. In a genetic construct the sequences of the tail of onemonoclonal antibody are replaced by the sequences from the leucine zipper region of Fos,and the sequences of the other monoclonal antibody are replaced by the sequences of theJun leucine zipper. The murine myoloma cell line Sp2/0 is transfected with these con-structs, resulting in the production of heterodimers of Other methods havefocused on obtaining single-chain bispecific molecules. These molecules consist of twovariable domains connected by a polypeptide spacer of two monoclonal antibodies,coupled by a linker (Traunecker et al., 1992).

Another approach to construct small bivalent antibody fragments is through thedimeric antibody fragments (diabodies) (Holliger et al., 1993). The diabodies can be eas-ily obtained from bacteria, can be expressed in high yield, and lack the Fc portions of thewhole immunoglobulin. These antibody fragments with two antigen-binding sites com-prise a heavy-chain variable domain connected to a light-chain variable domain on thesame polypeptide chain. Using a linker that is too short to allow pairing between the twodomains on the same chain forces the domains to pair with the complementary domains ofanother chain and create two antigen binding sites. It should be noted that antibody frag-ments are often preferable to complete antibodies, as the Fc region of antibodies can leadto undesirable targeting to cells expressing Fc receptors.

RECOMBINANT ANTIBODIES

A more recent strategy for cancer diagnosis and therapy is the application of geneti-cally engineered antibodies. This approach is logical because antibodies are the paradigmfor the design of high-affinity protein-based binding reagents. This technology includeschimeric and humanized antibodies, antibody libraries, and transgenic organisms as biore-actors (Gavilondo and Larrick, 2000). During the last 10–15 years, important advanceshave been made in the design, selection, and production of new types of recombinant anti-bodies. Recombinant antibodies have been reduced in size, dissected into minimal bindingfragments, and rebuilt into multivalent high-avidity reagents (Hudson, 1999).Recombinant antibody fragments have also been fused to radioisotopes and with a variety

Antigens and Antibodies 47

of molecules, including enzymes for prodrug therapy, toxins for cancer treatment, virusesfor gene therapy, cationic tails for DNA delivery, liposomes for improved drug delivery,and biosensor surfaces for cancer diagnosis and therapy for real-time detection of targetmolecules.

For clinical diagnostic applications, antibody fragments alone (without Fc) can pro-vide the full range of in vitro immunoassays through to in vivo tumor-targeting reagents.In fact, recombinant antibodies and their fragments have entered the clinic, both for can-cer diagnosis and for therapy. These reagents represent more than 30% of all biologicalproteins undergoing clinical trials for diagnosis and therapy. Clinical results confirm thatthese new antibodies, directed at the appropriate tumor markers (e.g., CD20 and HER-2),can control diseases without apparent side effects (Cragg et al., 1999). Innovative selectionmethods have enabled the isolation of high-affinity cancer-targeting and antiviral antibod-ies, the latter capable of redirecting viruses for gene therapy applications (Hudson, 1999).It is now possible to select high-affinity antibody fragments directly from a viral culturerather than from a live mouse. One significant advantage of this new technology is the iso-lation of antibodies with new binding specificities against hitherto refractory antigens, thusavoiding the limitations inherent in the mammalian immune response (De Haard et al.,1999). In addition, bispecific antibodies and related fusion proteins have been produced forcancer immunotherapy, effectively enhancing the human immune response in anticancervaccines and T cell recruitment strategies.

More recently, a novel technique has been developed for high-throughput screeningof recombinant antibodies, based on the creation of antibody arrays (De Wildt et al., 2000).This method uses robotic picking and high-density gridding of bacteria containing anti-body genes followed by filter-based ELISA screening to identify clones that express bind-ing antibody fragments. This approach can screen thousands of different antibody clonesat a time against a large number of different antigens. Thus, antibodies against impure pro-teins and complex antigens can be isolated. However, because a cellular extract containsthousands of different proteins, the detection sensitivity of this filter-screening techniquerequires considerable improvement to be useful for fingerprinting differentially expressedproteins.

The Food and Drug Administration has approved the use of engineered therapeuticantibodies. This technology is expected to be an important instrument in the toolbox of themolecular biologist. Although it is not exactly clear how monoclonal antibodies damagetumors in vivo, it is thought that the ability of the antibodies to crosslink membrane recep-tors and generate intracellular signals is part of the mechanism controlling the tumorgrowth.

ANTICANCER MONOCLONAL ANTIBODIES

Interest in the use of monoclonal antibodies in diagnosing and treating cancer hasundergone a resurgence during the last decade. The most important reasons for therenewed interest are (Murray, 2000): (1) the development of human or humanized anti-bodies through recombinant techniques, resulting in the decrease or elimination ofimmunogenicity, (2) approval of monoclonal antibodies (trastuzumab and rituximab) for

48 Chapter 2

cancer treatment by the Food and Drug Administration, and (3) antitumor effects of mon-oclonal antibodies, especially on solid tumors as shown by clinical trials when used aloneor in combination with chemotherapy or radiotherapy.

Trastuzumab (Herceptin; Genentech, South San Francisco, CA) is a humanizedmonoclonal antibody that recognizes the human oncoprotein HER-2/neu, which is over-expressed in some breast cancers and other tumors. Rituximab (Rituxan; Genentech,South San Francisco, CA, and I DEC Pharmaceuticals, San Diego, CA) is a geneticallyengineered chimeric monoclonal antibody containing murine light- and heavy-chain andkappa light-chain constant regions. It binds to CD20, a differentiation antigen found exclu-sively on B cells and on more than 95% of B-cell non-Hodgkin’s lymphoma, but not onhematopoietic stem cells, preB cells, normal plasma cells, or other tissues.

Monoclonal antibodies can mediate tumor destruction by both direct and indirectmechanisms. Direct mechanisms include (1) increased induction of apoptosis resultingfrom binding to calcium channels and (2) inhibition of ligand binding and suppressionof transcription factors within the tumor cells resulting from binding to growth factorreceptors (e.g., EGFR and HER-2). Monoclonal antibodies can also destroy tumor cellsindirectly through immunological mechanisms such as antibody-dependent cell-mediatedcytotoxicity and complement-dependent cytotoxicity (Murray, 2000). The type of antigento which the antibody binds is also relevant. The most effective monoclonal antibodies arethose that bind to specific receptors and possess the strongest biochemical and/or immuno-logical effects.

ANTIBODY CROSS-REACTIVITY

The ability of antibodies to bind molecules other than those molecules used as theimmunogens is well known. Such a binding is termed cross-reactivity. Cross-reactivity issometimes a source of confusion in the interpretation of immunohistochemically stainedpreparations because it cannot be detected by the usual controls for specificity.

Although the antigen-antibody reaction is highly specific, in some cases even a mon-oclonal antibody elicited by one type of antigen can cross-react with another type of anti-gen. In other words, two closely similar proteins may react with an antibody raised by onlyone of them. There are a number of reasons for cross-reactivity. This problem arises whenan antibody-combining site recognizes more than one antigenic determinant due tosimilarity in shape of different antigens. The cross-reaction can occur when the antigenicdeterminant site is a sequence of amino acids common to more than one antigen. Thiscommonality can occur when the antigenic determinant is conserved in a family of pro-teins. Many proteins possess homologies in their amino acid sequences and thus showimmunological cross-reactivity.

Cross-reaction can also occur when two different antigens share an identical epitopeor if antibodies specific for one type of epitope also bind to an unrelated epitope possess-ing similar chemical composition. However, the antibody-binding affinity for the cross-reacting epitope is usually less than that for the original epitope. Processing conditions canexpose an epitope related to the epitope under study but less so the former epitope. Carefulevaluation of a given monoclonal antibody and its determinant can be accomplished byepitope mapping (Nelson et al., 1997).

Antigens and Antibodies 49

Another reason for cross-reactivity is the use of less than pure protein or conjugatedor fusion proteins as immunogens; such immunogens produce a heterogenous populationof the antibody having considerable cross-reactivity to the contaminants (Javois, 1999).Cross-reactivities to the carrier protein to which the antigen has been conjugated or fusedcan be removed by affinity chromatography; however, a possible disadvantage of thismethod is that the most desirable immunoglobulins having the highest affinity bind thetightest and are difficult to recover. These problems can be prevented and increasedantibody specificity can be achieved by using synthetic peptides or protein fragments aseliciting antigens.

Another potential source of cross-reactivity is the presence of Fc receptors in cells ortissues, which bind the Fc region of the primary or secondary antibodies. These nonspe-cific sites can be blocked with normal serum or nonimmune immunoglobulins. When asecondary antibody is used for detection, the normal serum or immunoglobulin for block-ing should be from the same species as the secondary antibody.

Undesirable or nonspecific staining can also be the result of impure reagents used inthe staining. This background staining can be prevented by using purified reagents andoptimizing conditions for tissue processing including staining. Nonspecific binding canalso occur owing to ionic interactions with other proteins or organelles in the tissue(Grube, 1980). These interactions can be minimized by diluting the antibody and byincreasing the salt concentrations in the diluent and the rinsing solutions (Javois, 1999).

POLYREACTIVE ANTIBODIES

Polyreactive antibodies are naturally occurring antibodies. They are primarily fromIgM but also from IgG and IgA isotypes. Polyreactive antibodies are capable of reactingwith a variety of antigens that may differ among themselves. Unlike classic autoantibod-ies, which react with specific host antigens, polyreactive antibodies react with specific hostantigens. Polyreactive antibodies react with endogenous as well as exogenous antigens.The production of polyreactive antibodies is independent of antigen immunization.The affinity of these antibodies for different antigens varies but is relatively low comparedwith the affinity of monoclonal antibodies elicited by a mature antigen-driven response(Chen et al., 1995).

It is thought that polyreactive antibodies have physiological relevance. These antibod-ies probably are involved in defense distinct from their counterpart monoreactive antibodies.Polyreactive antibodies constitute a first line of defense against invading microorganisms byenhancing phagocytosis or complement-mediated lysis or by amplifying an ongoing specificantibody response. In other words, polyreactive antibody–producing/antigen binding cellsmight play a role in the development and maintenance of immunological tolerance. It is alsopossible that polyreactive antibodies play a role in the homeostasis of all internal biologicalsystems. These antibodies are known to bind to antigens in the blood and rapidly clear fromthe circulation (Sigounas et al., 1994).

Various assays, including ELISA, immunoblots, and chamber ELIspot, have beenemployed for demonstrating polyreactivity of preimmune antibodies in both mice andhumans (Ternynck and Avrameas, 1986; Quan et al., 1997; Klimman, 1994). In a complexbiological matrix, polyreactive antibodies are bound by components of the matrix, leaving

50 Chapter 2

only a small fraction of the antibody free in solution. This antibody is unmasked duringaffinity purification and becomes detectable by ELISA.

It is possible that limited denaturation of the antibodies occurs during purification,altering their monospecificity. For example, it has been demonstrated that the binding siteof M11, a murine, monoclonal antibody, is altered after purification at a low pH (2.2),resulting in its binding to many different proteins (McMahon and O’Kennedy, 2000). Theinference is that such a polyreactivity represents an acquired, artificial characteristic.

COMMERCIAL SOURCES OF ANTIBODIES

Antibodies are available from the following sources:

Affinity Research Products Ltd. (Exeter, U.K.)AMAC, Inc. (Westbrook, ME, U.S.A.)Becton-Dickinson (Mountain View, CA, U.S.A.)Biochemicals AG (Augst, Switzerland)Biogenesis, Inc. (Sandown, NH, U.S.A.)Biogenics (San Ramon, CA, U.S.A.)Biomedical Technologies (Stoughton, MA, U.S.A.)Bioproducts (Indianapolis, IN, U.S.A.)Biotrend Chemikalien GmbH (Cologne, Germany)Biozol Diagnostica GmbH (Eching, Germany)Boehringer Mannheim (Indianapolis, IN, U.S.A.)Cambridge Biosciences (Cambridge, U.K.)Calbiochem/Oncogene Research Products (Cambridge, MA, U.S.A.)Chemicon (El Segundo, CA, U.S.A.)Cis Bio International (Sur Yvette, France)Coulter Clone (Miami, FL, U.S.A.)Dako (Carpinteria, CA, U.S.A.; Glostrup, Denmark)Dianova (Hamburg, Germany)EuroDiagnostica (Amersfoort, The Netherlands)Fitzgerald (Concord, MA, U.S.A.)HistoCIS (Marseilles, France)Immunonuclear Corp. (Stillwater, MN, U.S.A.)Immunotech S.A. (Marseilles, France)Incstar (Stillwater, MN, U.S.A.)Labsystems (Chicago, IL, U.S.A.)LabVision/NeoMarkers (Fremont, CA, U.S.A.)Lipshaw (Pittsburgh, PA, U.S.A.)Loxo (Dossenheim, Germany)Monosan (Uden, The Netherlands)Neomarkers (Fremont, CA, U.S.A.)Nichirei Ltd. (Tokyo, Japan)Novocastra (Newcastle upon Tyne, U.K.)Ortho-Clinical Diagnostics (Amersham, U.K.)

Antigens and Antibodies 51

Pharmacia, Biotech (Uppsala, Sweden)Pharmigen (San Diego, CA, U.S.A.)Polysciences (Warrington, PA, U.S.A.)Progen (Heidelberg, Germany)QED Bioscience Inc. (San Diego, CA, U.S.A.)R & D Systems (Abingdon, U.K.)Rockland Inc. (Gilbertsville, PA, U.S.A.)Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.)Serotec (Oxford, U.K.)Transduction Laboratories (Lexington, KY, U.S.A.)Ultraclone (Isle of Wight, U.K.)Vector Laboratories (Burlingame, CA, U.S.A.)Wak-Chemie (Bad Homburg, Germany)Wako Chemicals GmbH (Neuss, Germany)Zymed Laboratories (San Francisco, CA, U.S.A.)

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Chapter 3

Fixation and Embedding

Because chemical fixation prevents cellular disintegration, it is the foundation for almost allmicroscopic studies. It is the most widely used method for preserving specimens for light andelectron microscopy. The reasons for its universal use are that it adequately preserves manycellular components, including soluble and structural proteins; prevents autolysis and dis-placement of cell constituents, including antigens and enzymes; stabilizes cellular materialsagainst deleterious effects of subsequent preparatory treatments, including incubations; andfacilitates conventional staining and immunostaining. Fixation also allows the clarity of struc-tural details shown by photomicrographs and electron micrographs, and it can be applied eas-ily. No other tissue preservation method can claim these advantages. Freezing is a usefuladjunct to chemical fixation. The biochemical reactions of formaldehyde and glutaraldehydewith proteins are discussed here and in more detail elsewhere (Hayat, 1986, 2000a).

An understanding of the effects of fixation on antigens and cell morphology is a pre-requisite to accepting the validity of immunohistochemistry. It is necessary to know theextent of antigen preservation or destruction due to fixation and dehydration-embedding.The preservation of antigenicity is adversely affected by any type of chemical fixationbecause epitopes may be masked sterically by the surrounding soluble proteins in the tis-sue fluid. The fixative may crosslink another molecule (usually a protein) directly to theepitope or in the vicinity of the antigen. The former may allosterically alter the epitopeconfiguration, inhibiting its recognition by the antibody. The latter may block the accessi-bility of the antibody to the antigen. In addition, the antigen may be degenerated by directcrosslinking with the aldehyde groups of the fixative. All of these changes may bereversible or irreversible or partly reversible or irreversible.

FORMALDEHYDE

Formaldehyde, a monoaldehyde, is preferred over glutaraldehyde (a dialdehyde)because the latter introduces mostly irreversible protein crosslinks, masking the epitopes.Formaldehyde penetrates rapidly into the tissue but crosslinks proteins slowly. Most ofthese crosslinks are reversible, and therefore masked epitopes can be easily unmasked bytreatments such as heating. However, for better preservation of cell morphology, a mixtureof formaldehyde (4%) and glutaraldehyde (0.01–0.1%) can be tried. Bouin’s fixative withits acidic pH should not be used.

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Reversibility of protein crosslinkages introduced by formaldehyde has been demon-strated in different biological systems. For example, formaldehyde has been applied at lowconcentrations as a reversible crosslinker for nearest neighbor studies of histone-histoneand DNA-histone complexes (Russo et al., 1981). It has also been shown that fixation ofpolyheads of the bacteriophage with 5% formaldehyde can be reversed up to 86% withacetic acid and sodium borohydride treatment (Baschong et al., 1983). This studywas carried out for stabilizing labile structures during both isolation and purification, fol-lowed by reverse fixation by acidification so that the protein constituents of these struc-tures can be characterized by gel electrophoresis. However, acid treatment may not allowthe recovery of the original protein structure in its native form of assembly.

Nature of Formaldehyde Solution

Aqueous formaldehyde as used for fixation contains mostly methylene glycol (~99%),its oligomers, and small amounts of formaldehyde. The proportion of the oligomers presentdepends inversely on the temperature. Formaldehyde solution cannot be obtained withoutthe formation of methylene glycol. It is not the formaldehyde molecule that is primarilyresponsible for rapid penetration into the tissue but methylene glycol, which is the majorcomponent of formaldehyde solution. At concentrations of 2% or less, the formaldehyde insolution is present practically only as the hydrated monomer

Methylene glycol is formed by the reaction between formaldehyde and water:

To maintain chemical equilibrium, more formaldehyde is found through the dehydrationreaction:

Elevated temperatures favor the dissociation of methylene glycol to formaldehyde duringfixation.

In addition to the small amount of formaldehyde originally present in the aqueoussolution, a little more is formed from the methylene glycol. However, formaldehyde com-ponent reacts very slowly with cellular proteins, and then it is slowly exhausted. Thismeans that the interior of the tissue block after fixation for 4–6 hr at room temperature isexposed mainly to methylene glycol; therefore, this portion of the tissue is fixed by ethanolduring dehydration, resulting in the coagulation of proteins.

Rapid and uniform fixation throughout the tissue block with formaldehyde can beobtained at high temperatures, for example, in a microwave oven. Such temperaturesenhance the speed and extent of formaldehyde reaction with proteins by dissociating themethylene glycol to formaldehyde as well as by depolymerizing the oligomers of methyl-ene glycol (Boon et al., 1988).

Mechanism of Fixation with Formaldehyde

Like glutaraldehyde, formaldehyde is an additive fixative used for preserving tissuesfor light microscopy (Hayat, 2000a). When formaldehyde reacts with an amino acid that

Fixation and Embedding 55

has an available hydrogen site, a reactive hydroxymethyl group is produced, which is theaddition product. Subsequently, a second hydrogen site may react with this addition prod-uct, yielding a methylene bridge between two amino acids in the protein. As a result of thisinteraction a large number of hydroxymethyl groups are produced, utilizing reactivespecies on the side chains of amino acids or directly from the peptide bond (Table 3.1). Inother words, additional reactions of formaldehyde with the hydroxymethyl groups and pre-viously unreacted amino acid side chains form the methylene bridges that are the proteincrosslinks of formaldehyde fixation. Formaldehyde reacts through several steps to form amethylene bridge between two neighboring amino groups of amino acids. Neutral pHfavors the formation of such bridges.

The primary reason for using neutral-buffered formalin is that at this pH hydrogensites in peptide molecules are available for linkage because they are in an uncharged state.In contrast, an acid pH induces formation of charged amino groups that lack reactivehydrogen sites. Nonscientific reasons for using formalin are that it is inexpensive, and eas-ily available, and diagnostic pathologists and technicians have been trained to use it.

Formaldehyde introduces both intramolecular and intermolecular crosslinks betweenproteins involving hydroxymethylene bridges, which change the three-dimensional struc-ture of proteins. Such changes involve the tertiary and quaternary structures of proteins,whereas the primary and secondary structures are little affected. It has been shown that thesecondary structure of purified protein molecules remains mostly unaltered during fixationwith formaldehyde (Mason and O’Leary, 1991). Even when the quaternary structure ischanged by formaldehyde fixation, the secondary structure can remain intact.

Formaldehyde does not react with all functional groups in proteins with equal rapid-ity; it reacts first with lysine and cysteine, and subsequently these amino acids are

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crosslinked to glutamine, asparagine, and arginine. This does not mean that the fixativealters the amino acid sequence of the antigen molecule; the structure of the epitope ismaintained even though it may be masked. However, various proteins respond differentlyto fixation with formaldehyde. It is relevant to point out that aldehyde fixation does notcompletely abolish the selective permeability of cell membranes (Hayat, 1986). Therefore,it can be inferred that membrane proteins and other proteins are not destroyed by fixationwith formaldehyde.

The strongest indirect evidence in support of the role of protein crosslinking in creat-ing a barrier to the penetration of antibodies and their reaching to the epitopes emergesfrom the crosslinking ability of glutaraldehyde. It is known that this dialdehyde introducesextensive protein crosslinks that are mostly irreversible, which significantly inhibits anti-genicity (Hayat, 1986, 2000a). This inhibition is due to the crosslinking of protein antigensas well as crosslinking and compacting of proteins surrounding the antigen molecule. Mostepitopes in most protein antigens studied so far are located at or near the exposed surfaceof the antigen molecule. Therefore, compacted proteins surrounding the antigen moleculewill prevent the recognition of epitopes by the antibody. The small size of the epitopesmakes them susceptible to masking by the surrounding crosslinked proteins.

Comparison of Formaldehyde with Glutaraldehyde

The effect of aldehydes on tissue proteins is exceedingly complex. Numerous factorsinfluence the immunostaining of epitopes, and the fixation of the tissue with aldehyde isthe most important parameter. Fixation with an aldehyde results in the crosslinking of pro-teins, including antigens, resulting in their stabilization, protection, and anchoring in situ.Crosslinking protects the modified antigens from denaturation at elevated temperatures. Itis known that the use of high temperatures, for example from a microwave oven, may per-manently denature unfixed antigens (epitopes), whereas similar temperatures have a min-imal adverse effect on aldehyde-fixed antigens. Studies of unfixed, purified proteinsindicate that they show denaturation transitions in the 70–90°C temperature range, whereassuch proteins do not exhibit these changes at the same temperature when they have beenplaced in formaldehyde solutions (Mason and O’Leary, 1991). The absence of phasetransition is thought to be due to stabilization of the protein structure by formaldehydetreatment.

Glutaraldehyde or formaldehyde alone or together are most commonly used to stabi-lize proteins. Both have advantages and limitations, as explained below (Hayat, 1986,1999). Glutaraldehyde molecule is a dialdehyde with two aldehyde moieties, one on eachend of a straight hydrocarbon chain.

Fixation and Embedding 57

The presence of the two aldehyde groups is the reason for its being the most effectiveprotein crosslinking molecule. Glutaraldehyde introduces both intramolecular and intermol-ecular protein crosslinks that are predominantly irreversible during subsequent processing,including incubations of the tissue. This versatile property results in excellent preservationof the ultrastructure; however, it also has limitations. The dialdehyde produces strong, irre-versible protein crosslinks when used in standard concentrations (2–3%). Therefore, itmasks most epitopes. In addition and more important, steric hindrance resulting fromextensive crosslinking of cellular proteins in general inhibits the antibodies from reachingthe intracellular epitopes. Thus, glutaraldehyde is less suitable for light and electron micro-scopic immunocytochemistry. Some hardy antigens that are less susceptible to the effect ofaldehydes, however, can be studied following fixation with glutaraldehyde alone or in com-bination with formaldehyde for electron microscopic immunocytochemistry.

For light microscopic immunohistochemistry and immunocytochemistry, formaldehydeis preferred over glutaraldehyde because it is a monoaldehyde and thus penetrates thetissue more quickly but crosslinks proteins more slowly than glutaraldehyde, a dialdehyde.Formaldehyde does not react with all the functional groups in proteins with equal rapidity.In its reactions with proteins, the first step involves the free amino groups with the formationof amino methylol groups, which then condense with other functional groups such asphenol, imidazole, and indole to form methylene bridges The occurrence of thesebridges is considered responsible for the fixation of proteins by formaldehyde underconditions of fixation.

The process of masking is progressive, i.e., the longer the fixation, the stronger thecrosslinking as well as epitope masking. It is well established that with increasing dura-tions of fixation, antibodies show a nearly linear decrease in immunostaining, indicating asuccessive masking of epitopes (Werner et al., 1996). Therefore, the duration of fixationshould be taken into account while determining the duration of treatment for epitoperetrieval. Generally, mild fixation with formaldehyde is preferred. Some epitope types arenot affected by standard or prolonged fixation with formaldehyde or a mixture offormaldehyde and glutaraldehyde or, as stated earlier, glutaraldehyde alone.

The advantage of using a mixture of formaldehyde (4%) and glutaraldehyde (~0.1 % ora lower concentration) is improved preservation of cell structure. Although formaldehyde isbetter than many other fixatives for preserving morphological details, it is far inferior to glu-taraldehyde. The quality of morphological preservation with glutaraldehyde has been com-pared with that obtained with formaldehyde (Hayat, 2000a). These studies clearly indicatesuperior structural preservation with glutaraldehyde. In fact, fixation with formaldehyde isunacceptable for routine electron microscopy, which demands superior structural preserva-tion because of its high resolving power. However, for most electron microscopic immuno-cytochemical studies, a mixture of formaldehyde and glutaraldehyde is recommendedbecause many types of epitopes are irreversibly masked when the latter alone is used.Although at present formalin is routinely used for the fixation of surgical tissue specimens,the advantage of a mixture of formaldehyde and glutaraldehyde cannot be overemphasized.

Fixation with Formaldehyde

There is no optimal universal fixative for all types of antigens, so the choice of a fix-ative depends on the type of epitope and the tissue under study. Furthermore, the selection

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of a fixative is always a compromise between the preservation of antigenicity and cell mor-phology. Although the best fixative for a specific epitope is determined by trial and erroror by recommendations from a published study, 4% formaldehyde in 0.1 M buffer (pH 7.2)is recommended for adequately preserving both the antigenicity and cell morphology forimmunostaining. Alternatively, 10% neutral-buffered formalin can be used. Standard fixa-tion for 4–6 hr at room temperature is considered a mild and effective fixation.

Most of the reactions of this aldehyde with proteins during mild fixation arereversible, and the resultant protein crosslinks for the most part can be weakened and/orbroken with treatments such as heating. Unbound formaldehyde is also removed duringwashing. The reactions of formaldehyde with proteins are influenced by several factors,including its concentration, pH, temperature, and duration of fixation. In general, highervalues of these parameters result in increased binding of formaldehyde. The maximumbinding occurs at pH 7.5 to 8.0.

However, it should be noted that the fixation throughout the tissue block is completedin ~24 hr, but pathological specimens are usually fixed for less than this duration beforetheir further processing. As a result, the tissue is only partially fixed with the aldehyde, andits fixation is completed with the dehydrating ethanol. It means that the tissue is fixedpartly by protein crosslinking with the former and partly by coagulation with the latter(Battifora, 1991). Consequently, the specimen may show heterogenicity of immunoreac-tivity; different areas of the section may exhibit different intensity of staining. Variablestaining density in different areas of the tissue block may also be caused by the presenceof air bubbles in the vial containing the specimens during fixation. Tissues fixed with coag-ulating fixatives such as ethanol generally do not benefit by antigen retrieval treatments.

Although 10% neutral buffered formalin is the most commonly used fixative andyields satisfactory results, the optimal preservation of certain antigens requires a differentfixative. Three examples are given. The immunostaining of transforming growthand thrombomodulin in the human skin has been reported to be superior in the specimensfixed with methanol-Carnoy’s solution (methanol: chloroform: acetic acid, 6:3:1) com-pared with that in the formalin-fixed specimens (James and Hauer-Jensen, 1999). Optimalduration of fixation is 24 hr. Similarly, the antigenicity of the proteinase-K–resistant formof the prion protein in brain tissue is better preserved in the Carnoy-fixed specimens(Giaccone et al., 2000). Another example is leukocyte antigenicity in various rat tissues,which is better preserved in the Carnoy-fixed specimens (Shetye et al., 1996).

Effect of Prolonged Fixation with Formaldehyde

Why does prolonged fixation with formaldehyde cause progressive loss ofimmunoreactivity? The reasons are fairly clear. Formaldehyde, being a small molecule

penetrates readily and adds onto protein molecules, forming mostly reversiblecrosslinks. However, the crosslinking occurs slowly because formaldehyde contains onlyone aldehyde group, and it takes time to align a molecule(s) to accomplish crosslinking.Thus, reversible crosslinks and loosely bound fixative molecules are mostly removed dur-ing antigen retrieval processing, resulting in satisfactory immunoreactivity. As the time offixation increases, additional stronger crosslinkages are formed, which in turn cause pro-gressive loss of immunoreactivity. Such strong crosslinkages may not only alter the

Fixation and Embedding 59

conformation of the antigen molecule but also create a barrier to antibody access to theepitope. The formation of extensive crosslinkages during prolonged fixation for weeks ormonths is supported by the observation that such tissue blocks become hard and difficultto section. It is well known that archival tissues are difficult to section.

The effect of prolonged fixation with formaldehyde on the antigenicity of the nucleusmay differ from that of the cytoplasm. This phenomenon is exemplified by Bcl-2 and Bax,members of the same family of proteins involved in apoptosis regulation; these proteinsreside in the cytoplasm as well as in the nucleus. It was recently demonstrated that pro-longed fixation with formaldehyde alone irreversibly reduced nuclear or mitotic Bcl-2immunoreactivity even after heat-mediated antigen retrieval in monolayers of MCF-7human breast cancer cells (Hoetelmans et al., 2001).

Heat treatment, on the other hand, elevated cytoplasmic immunoreactivity of Bcl-2.However, nuclear and mitotic Bcl-2 immunoreactivity was clearly present when these cellswere fixed with formaldehyde (3.6%), followed by postfixation with methanol for 10 minat –20°C. Treatment with ice-cold methanol makes the cell membrane permeable, allow-ing antibody access to intranuclear antigens without protein relocalization. Extensive pro-tein crosslinking with formaldehyde is required for maintenance of intranuclear Bcl-2immunoreactivity. In contrast to Bcl-2, Bax immunoreactivity was detected in nuclear andcytoplasmic compartments regardless of the duration of formaldehyde fixation used.

In light of the aforementioned information, when tissue specimens are exposed toformaldehyde for longer durations due to unavoidable circumstances, immunohistochem-ical findings should be interpreted with caution, as many tissue antigens could be lost orirreversibly masked.

Formalin Substitute Fixatives

Because formalin poses occupational hazards, a number of ethanol-based fixativeshave been introduced, some of which are commercially available. These substitutes for for-malin include Notox, Omnifix, Stat Fix, Histochoice, Tissue-Tek, F13, Carnoy, Bouin fluid,and methacarn. Although these fixatives have been recommended for histology laboratories,in comparison with formalin they have limitations. Many of these formalin substitutes arecoagulating fixatives which precipitate proteins by breaking hydrogen bonds in the absenceof protein crosslinking. This limitation results in inadequate cellular preservation. Thesesubstitutes also cause cellular shrinkage and brittleness. The inclusion of polyethyleneglycol or acetic acid is thought to minimize these harsh effects (Warmington et al., 2000).

Another drawback of some of these formalin substitutes is that they tend to cause an arti-factual shift in the intracellular immunoreactivity. Such a shift from the cytoplasm to thenucleus has been demonstrated for growth factor peptides (Bos et al., 2000). The result of sucha shift is false nuclear signal dominance or exclusive nuclear staining. This shift is thought tobe owing to electrostatic interaction between ligands and DNA or other nuclear components,as fixation (anchoring of cell components) remains incomplete with alcohol-based fixatives.Furthermore, these fixatives make the nuclear membrane permeable, allowing bidirectionalmovement of ligands between the cytoplasm and the nucleus. In conclusion, the chemistry offormalin fixation is well known, whereas this is not true for the formalin substitute fixatives.Therefore, these substitutes cannot be recommended for immunohistochemistry.

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Another fixative, Kryofix (E. Merck, Darmstadt, Germany) has been recommendedas a replacement for formaldehyde in immunohistochemistry by Boon and Kok (1994).Kryofix is a coagulant fixative containing 50% ethyl alcohol and polyethylene glycol (mol.wt. 300). Both ethyl alcohol and polyethylene glycol diffuse rapidly into the tissue, and thetissue fixation is completed in Kryofix in 90 sec with microwave heating. I do not havepersonal experience with Kryofix.

Fixation Conditions

Some major factors that adversely affect immunostaining are delays in transferringthe tissue blocks into the fixative after their surgical removal as well as shorter or longerthan optimal duration of fixation. Any delay in the exposure of the tissue to the fixativeinvites increasing proteolytic degradation of antigenicity. Therefore, if possible, surgicaltissues must be placed directly into the fixative immediately after their removal. If delay isunavoidable, the tissue can be refrigerated prior to fixation.

It is not uncommon for thick (~5 mm) surgical tissues excised for diagnostic pathol-ogy to be underfixed with formalin, especially the core of the tissue block. As an average,fixation in formalin solution for less than 24–48 hr, depending on the size of the tissueblock, tends to crosslink only the periphery of the specimen. Under this condition, the coreof the tissue block either remains unfixed or fixed by coagulation with the alcohol usedsubsequently for dehydration. Sections cut from the core tend to show autolysis and inad-equate immunostaining, resulting in false-negative staining. Such inadequate staining canbe improved by attaching paraffin sections to glass slides and then removing the paraffinwith an organic solvent (Eltoum et al., 2001). This treatment is followed by rehydration,buffer rinse, and refixation with formalin. The fixative is removed by rinsing with bufferbefore staining. If necessary, incompletely fixed, paraffin-embedded tissues can bedeparaffinized and refixed and reembedded, risking some damage to cell morphology andimmunogenicity. These corrective methods become useful only when additional tissuespecimens are not available.

Overfixation of specimens may also be encountered, resulting in weak or absentimmunostaining, depending on the susceptibility of a specific antigen to the fixative. Asalready emphasized, prolonged fixation introduces excessive protein crosslinking, whichhampers antigen accessibility to the antibodies. In addition, 10% formalin solution containsonly 4% formaldehyde, and the remaining components may damage antigens during pro-longed fixation. If prolonged fixation has been carried out, immunostaining can be increasedby the following steps: robust antigen retrieval by heating, higher antibody concentrations,longer durations of incubations in the reagents, and signal amplification. To determine theoptimal increase in heat or protease treatment to counteract the effects of prolonged fixation,three slides can be processed with progressive doubling of the duration of treatment (Werneret al., 2000). The best stained slide of the three is used for interpreting the results of the study.However, some of these approaches may increase the background noise.

Overfixation of specimens is also recognized by difficulty in sectioning because ofexcessive hardness of the tissue. This problem arises when tissues are fixed with formula-tions containing ethanol, methanol, or acetone. Excessive dehydration with an organic sol-vent may also cause tissue hardness, especially of small specimens (1–2 mm). Such

Fixation and Embedding 61

specimens may shatter instead of being sliced during cutting. The increased hardness canbe prevented by shortening the duration of dehydration. If the tissue has been excessivelyhardened, it can be partially corrected by briefly soaking in water (Eltoum et al., 2001).Water tends to penetrate up to 0.5 mm into the tissue block. A large tissue block can be cutinto small pieces before soaking in water. Overfixation with protein crosslinking aldehy-des can also be partially reversed with washing in water or aqueous buffers.

EFFECT OF HEATING ON FIXATION WITH GLUTARALDEHYDE

No other fixative surpasses glutaraldehyde in preserving cellular details, provided thespecimen size is very small. Homogenous, irreversible protein crosslinking is the reasonfor the superior fixation with this dialdehyde. However, glutaraldehyde penetrates thetissue block rather slowly for three reasons: (1) it is a relatively large molecule, (2) rapidprotein crosslinking in the outer layers of the tissue block hinders its deeper penetration,and (3) because fixation is carried out at a slightly alkaline pH, the presence of glu-taraldehyde oligomers is promoted, and they do not penetrate the tissue as rapidly as thefree monomer dialdehyde.

Rapid penetration of the fixative avoids postmortem alteration in the tissue specimen.The speed of glutaraldehyde penetration (diffusion) can be increased at higher temperatures,provided the duration of fixation is short. Diffusion increases exponentially as temperaturerises. Diffusion of glutaraldehyde can be increased in the presence of microwave heating.Compared with other heating systems, microwave heating increases the temperature rapidlyand uniformly throughout the tissue block. There are several reasons for such acceleration ofglutaraldehyde diffusion, which are explained below.

Microwave heating causes depolymerization of glutaraldehyde into small monomers,enhancing the fixative penetration as well as acceleration of its reactions with amine groups.Microwave heating also induces changes in the tertiary structure of proteins, exposingreaction sites that remain unexposed at room temperature for reaction with glutaraldehyde(Kok and Boon, 1990). It is also known that microwaves accelerate the diffusion of polarcompounds in the presence of concentration gradients. Because both water and glu-taraldehyde solutions are polar, they are capable of absorbing energy while being heatedwith microwaves. Consequently, fixation with dialdehyde in a microwave oven can beaccomplished in seconds or minutes for light and electron microscopy.

As stated above, monomeric glutaraldehyde diffuses rapidly compared with its poly-mers. Heating increases absorption at 280 nm, which is the purification index for monomeicglutaraldehyde (Ruijgrok et al., 1990). Monomeric glutaraldehyde diffuses faster than do itsoligomers. However, longer durations of heating tend to produce significant amounts ofalpha and beta unsaturated polymers, which exhibit absorption at 235 nm.

Microwave heating permits the use of glutaraldehyde at very low concentrations,which still yield good ultrastructural preservation and superior antigen preservation.Fixation with glutaraldehyde in a microwave oven also minimizes tissue shrinkage.Moreover, microwave heating prevents some of the artifacts formed during glutaraldehydefixation. A well-known example is the presence of parathyroid cell variants thatare regularly seen in immersion-fixed specimens. These variants can be avoided with fix-ation by vascular perfusion, however, in certain cases vascular perfusion is not possible.

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A better alternative involves glutaraldehyde fixation by heating in a microwave oven. Thisapproach completely prevents the artifactual parathyroid cell variants (Marti et al., 1987).The fixation in this case is uniformly accelerated throughout the specimen block. Someevidence indicates that microwave heating followed by osmication enhances postfixationwith this metal.

However, fixation with glutaraldehyde at standard concentrations (2–3%) with orwithout microwave heating is not recommended for immunohistochemical and immuno-cytochemical studies, except for localizing epitopes that are resistant to this dialdehyde.The reason for this nonrecommendation is that most epitope types become irretrievableafter fixation with glutaraldehyde. Nevertheless, when the quality of ultrastructural preser-vation has an equal priority with that of immunolabeling, glutaraldehyde can be used atvery low concentrations in a mixture containing 2% formaldehyde. For example, cellsurface epitopes of rat mast cells were preserved by using this procedure (Jamur et al.,1995). The cells were fixed with a mixture of 2% formaldehyde and 0.05% glutaraldehydecontaining 0.025% in 0.1 M cacodylate buffer (pH 7.4) for 4 sec at 100% power ina 550-W microwave oven.

As explained in this chapter, glutaraldehyde introduces mostly irreversible proteincrosslinks that may alter the conformation of the antigen (epitope) molecule. Such exten-sive crosslinkages become a barrier to the antibody penetration, and thus its accessibilityto the epitope is hindered. This impediment becomes a serious problem when monoclonalantibodies specific for only one epitope type are used and/or when epitopes are not locatedat the surface of the antigen molecule.

As explained earlier, large polymers of glutaraldehyde are formed in the outer layersof the tissue block, which impede further penetration of the fixative into the core of thetissue. Such an impediment is due to steric hindrance and/or formation of nucleation sitesto which fixative molecules may attach. The latter will consume large quantities of glu-taraldehyde, depleting its concentration in the solution. The net result will be unevenfixation of the tissue block. The uneven fixation, however, can be avoided by providingconditions that initially allow an adequate penetration of glutaraldehyde throughout thetissue block, followed by the formation of crosslinks and polymers. This can be accom-plished by presoaking the specimen in a low concentration (0.5%) of glutaraldehydeat 0–4°C, followed by microwave heating of the fixative at 45°C (Ruijgrok et al., 1993).The durations of these two steps are determined by trial and error.

The initial cold temperature slows the formation of polymers in the outer cell layersof the tissue, allowing glutaraldehyde penetration. An instant homogenous heating in amicrowave oven facilitates uniform protein crosslinking throughout the tissue block afterthe fixative has fully penetrated. Other types of heating do not impart an even rise in tem-perature throughout the tissue block.

MICROWAVE HEAT–ASSISTED FIXATION WITHOSMIUM TETROXIDE

Standard chemical fixation fails to preserve extracellular materials. In contrast,heating or the high-pressure freezing-freeze-substitution technique is

able to preserve such materials (Eggli and Graber, 1994). The former technique is simpler

Fixation and Embedding 63

and cheaper and can be completed in a conventional microwave oven with a maximalpower output of 650 W or higher. A water load (200 ml in a beaker), placed in a rear cor-ner of the oven, serves as a damper to increase heating times to 10 sec or longer. Tissuespecimens (e.g., nervous, ocular, and skin tissues) are fixed for 2 min at room temperaturewith 2% in 0.2 M sodium cacodylate buffer (pH 7.2, 450 mOsm). They are trans-ferred into small glass vials containing 10 ml of the fixative. The vials are placed inthe center of the microwave oven and heated at maximal power output and at a frequencyof 2,450 MHz until the temperature between 43°C and 40°C is attained (~12 sec). Thespecimens are removed from the oven and kept at room temperature for 10 min prior towashing in 0.1 M buffer and embedding in an epoxy resin. The results of this procedureare shown in Fig. 3.1.

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ROLE OF MICROWAVE HEATING IN ENZYME CYTOCHEMISTRY

Typically, residual enzyme activity is localized at the subcellular level after fixationwith an aldehyde followed by incubations. Aldehydes, especially glutaraldehyde, denatureenzyme molecules to various degrees. Lengthy incubations under nonphysiological condi-tions may cause the loss of structural details. To improve preservation of both the enzy-matic activity and the ultrastructure, incubation durations can be shortened undermicrowave heating (Rassner et al., 1997). The advantage of incubation under microwaveheating becomes apparent when one considers that it allows incubation of tissuespecimens, whereas conventional incubation requires tissue slices of 0.1 mm.

Tissue specimens are fixed for 16 hr with a mixture of 2% paraformaldehydeand 2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) containing 0.06% calciumchloride. After rinsing in buffer, the specimens are placed in 10-ml snap-cap glass vialscontaining 2 ml of incubation medium. The vials are capped to avoid contamination ofthe incubation medium from the water bath. They are placed into a staining jar (3 × 3.5 ×2.5 inches) which has been filled with 150 ml of tap water at 22°C. The microwave incu-bations are carried out twice for 30 sec each in a microwave oven at the highest power set-ting (900 W at 2,450 MHz, with two water loads 250 ml each) placed in the rear of theoven. The temperature in the oven is not allowed to exceed 40°C.

The water of the water bath is changed between the two 30-sec pulses. The tempera-ture of the water bath rises during microwave heating from 22°C to 40°C. The stainingjar is removed from the oven, and the incubation is continued for an additional 30 min at37°C. After washing with distilled water, the specimens are postfixed with a mixture of 1%

and 1.5% potassium ferrocyanide in 0.1 M cacodylate buffer (pH 7.2) for 90 min inthe dark at room temperature. The specimens are rinsed in buffer and then immersed in 2%aqueous uranyl acetate. Detailed methods for achieving accelerated visualization of acetyl-cholinesterase activity at motor endplates using microwave heating have been presented byPetrali and Mills (2001).

Fixation for Enzyme Cytochemistry Using Microwave at RelativelyLow Temperature

The activity of some enzymes can be preserved by tissue fixation with aldehydes ina microwave oven at a low temperature for electron microscopy. This procedure has beenused for observing cytochrome oxidase in mitochondria in hamster submandibular glandtissue (Moriguchi et al., 1999). The activity of this enzyme has also been studied in the iso-lated mitochondria of this tissue, using the same method (Moriguchi et al., 1998). Thisprocedure also has been employed for confocal laser scanning microscopy of enzymaticactivity (Moriguchi et al., 1999). The following protocol was used for processing the tis-sue for electron microscopy.

The microwave processor is fitted with a thermometer, timer, stirrer, and power levelcontroller (150 W to 400 W) (MI-77, Azumaya Company, Tokyo, Japan). One glass Petridish with 70-ml capacity containing 50 ml of a mixture of 2% paraformaldehyde and 0.5%glutaraldehyde containing 45 mg/ml sucrose in 0.1 M PBS (pH 7.4) is placed in a largePetri dish containing 250 ml of chilled water in the processor. The specimens are heated ina microwave oven for 10 min at ~4°C at a low energy level of 150 W.

Fixation and Embedding 65

CRYOPRESERVATION IN THE PRESENCE OF MICROWAVE HEATING

Cryoinjury to the specimen is caused directly by extra- or intracellular ice crystal for-mation as well as by ice-induced solution effects during cryopreservation. Ice crystals seri-ously deform cell components. Another disadvantage of the formation of ice crystals nearthe specimen surface is slowing the cooling rate in areas below the surface because theirthermal conductivity is about half that of solid water in a noncrystalline state. Furthermore,ice crystal formation is accompanied by the generation of latent heat, which also slowsdown the freezing rate.

Apparently, cryoinjury can be avoided by eliminating ice crystal formation and vitrify-ing the specimen. Vitreous state can be achieved by ultrarapid cooling (> ) or usinghigh concentrations of a cryoprotectant. However, the former is difficult to attain, and the lat-ter tends to cause chemical toxicity and high osmotic stress. Because biological specimenspossess low thermal conductivity and high thermal capacity, ultrarapid cooling can beobtained only for very small specimens. The above-mentioned difficulties in obtaining ultra-rapid freezing can be minimized in the presence of microwave heating. This treatment sup-presses ice crystal formation near the specimen surface, thereby extending the depth of goodfreezing from the specimen surface. Another advantage is better reproducibility of resultsbecause the state of water near the specimen surface is under control with microwave heating.

Two mechanisms responsible for decreased rate of ice crystal growth are suggested.It is possible that the electric field component of electromagnetic radiation interacts withdipolar water molecules, disrupting the ice nucleation phenomenon (Hanyu et al., 1992).In other words, microwave heating reduces the size and number of ice crystal nucleationcenters near the specimen surface. An alternative explanation is based on microwave radi-ation interfering with the kinetic processes of ice crystal growth (Jackson et al., 1997). Foran ice crystal to form and grow, each water molecule must have an appropriate spatial ori-entation, position, and energy. Rapid ice crystal growth requires the molecular clusters toshare edges and faces with the ice lattice without the induction of mutual strains. Thetorques produced by a microwave field can increase the number of available isomeric con-figurations, reducing the likelihood of a cluster of molecules having a configuration suitedto integrating into a crystal lattice. Further development in the application of microwaveheating to vitrification of biological specimens is awaited.

Two apparatuses have been constructed for achieving ultrarapid freezing in the pres-ence of microwave heating (Hanyu et al., 1992; Jackson et al., 1997). Microwave treatmentcan be employed with or without a cryoprotectant. According to one method, microwaveheating is used at 2.45 GHz for a short duration (50 msec) immediately before and duringtissue contact with the surface of a copper block cooled with liquid nitrogen (Hanyu et al.,1992). The ultrastructure is well preserved to a depth of from the contact surface,which is comparable to the depth obtained by the metal contact method using liquidhelium in the absence of microwave heating.

PARAFFIN EMBEDDING

For routine immunohistochemestry, surgical and other tissues are embedded in paraf-fin which is a mixture of hydrocarbons. Automated paraffin tissue processors are com-mercially available that customize the schedule to meet specific needs. However, tissues of

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various types and sizes as well as the particular study objective require optimal conditionsof dehydration, infiltration, and embedding in paraffin. Chemical and physical changesoccur in specimens during these treatments, affecting the sectioning and immunostainingqualities. Longer than optimal durations of these steps is a common habit, resulting in hardand brittle tissues that are difficult to section. It should be noted that additional fixationoccurs during dehydration, accompanied by antigen masking and lipid dissolution.

After fixation, a series of ethyl alcohol (a water-miscible solvent) of ascending con-centrations is used to remove water from the tissue. Free water from the tissue is easilyremoved by diffusion. Water attached to the tissue by hydrogen bonds is also replaced byethyl alcohol of higher concentrations. The efficacy of a solvent depends on its hydrogenbonding strength and molecular weight (Wynnchuk, 1993). Higher temperatures, vacuum,and microwave heating expedite the speed of dehydration, allowing shorter durations ofdehydration.

Xylene (an aromatic hydrocarbon) is used to replace ethyl alcohol from the tissuebefore infiltration with paraffin. Xylene is miscible with ethyl alcohol and paraffin. Xyleneis called a clearing agent because it has a refractive index similar to that of proteins andthus renders tissue more or less transparent. It is generally satisfactory when the tissueblocks are not thicker than 3–4 mm. Xylene must be completely removed with paraffin,otherwise tissue will not section. Excessive exposure to xylene causes further denaturationof tissue proteins, causing difficulties in sectioning. Some lipid extraction also occurs inthe presence of xylene. The treatments mentioned above to expedite the diffusion of ethylalcohol also speed up the penetration of xylene. Xylene is also used between ethyl alcoholand mounting sections with resinous mounting medium after staining. The volatility andinflammability of xylene render it potentially dangerous. It must be used in a fumehood.

While tissue is in xylene, gradual infiltration with paraffin is carried out. For tissuesof a small size, 2 to 3 hr of paraffin infiltration is adequate. For large tissues (5–10 mm),overnight infiltration is required. The temperature during infiltration must not be higherthan 4° above the melting point of paraffin (54–58°C).

Vacuum embedding can be carried out to remove air bubbles from the tissue and rap-idly replace the clearing agent with paraffin. This approach is especially desirable for air-containing tissues such as lung or hard tissues such as fibrous or scar tissues. The vacuumshould not exceed 400–500 mm of mercury to avoid damage to the tissue.

Paraffin blocks are trimmed with a scalpel, a razor blade, or a hot spatula andmounted on wooden or fiber blocks. While being sectioned, longitudinal block edges mustbe parallel to the knife edge to obtain a ribbon. Paraffin sections of any thickness showcompression, which is usually relieved when they are floated on a glass slide and dried.

It should be noted that the melting point and crystalline structure of paraffin influencethe section quality. Paraffin of a lower melting point is less brittle when solidified; how-ever, it tends to show more compression during sectioning. On the other hand, paraffin ofa higher melting point provides a better support for hard specimens. Paraffin of a smallercrystalline structure adheres closely to the cell components in the embedded tissue, pro-viding good support for sectioning. Paraffin sections of a larger crystalline structure showmore pronounced curvature, which is difficult to flatten after sectioning. To obtain crystalsof a small size, paraffin should be cooled rapidly. Deeper layers of the tissue block containlarger and looser crystals, resulting in poor quality of sections in these layers.

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Paraffin Embedding in Microwave Oven

Recently, a new type of microwave oven (HFX-800, Meditest, Illatos ut 9, 1097Budapest, Hungary), combining microwave heating and vacuum, was introduced (Kovacset al., 1996). The oven is reported to allow fixation, dehydration, paraffin embedding, andsection staining. The histoprocessing is completed in depending on the thick-ness of the tissue block. Decalcification of the bone specimen can also be carried out inthis oven.

The power level can be switched from 10 to 650 W by the chosen cycle time.The temperature can be preset and subsequently controlled automatically between 0 and120°C. Vacuum is generated by a built-in vacuum pump (operating at 12 V), producing0.0–0.3 bar vacuum. The vacuum level is indicated by an automatic meter. Since the ovenprovides a sealed and ventilated system, the evaporation of formalin, ethanol, isopropanol,and other reagents does not affect the operator. Vapors and fumes are extracted by contin-uous ventilation. The oven weighs about 21 kg. Its use is awaited.

Paraffin Embedding in Vacuum-Microwave Oven

Vacuum combined with microwaving has been tried for embedding the tissue inparaffin, using Milestone’s MicroMED LAVIS-1000 machine (Marani et al., 1996; Boschet al., 1996). The advantage of this system is that microwaves travel with ease through avacuum, whereas conventional heating under vacuum is difficult. This machine providespressure reaching 100 hPa, and the microwave oven attains a maximum power of 1,000 W;its cycle time can be adjusted between 0.1 and 0.5 sec. The machine is equipped with aninfrared temperature probe which allows temperature control from outside the unit.

To obtain satisfactory results, coordination of temperature with vacuum is necessary.Paraffin embedding is carried out in a stepwise descending series: 700 hPa, 500 hPa,300 hPa, and 100 hPa. A too-rapid lowering of the pressure is damaging to tissue mor-phology. The temperature during dehydration with isopropanol should not exceed 60°C.Further improvements of this system are awaited.

Microtomy of Paraffin-Embedded Tissues

Commercial paraffin is a mixture of a straight chain of hydrocarbons that containadditives. Both the melting and the plastic points of paraffin are related to the sectioningproperties. The plastic point occurs ~10°C below melting point. The role of the meltingpoint becomes apparent when one considers that the higher-melting-point hydrocarbonscrystallize first as flat plates that accumulate on one another as successively lower-melting-point hydrocarbons crystallize (Allison, 1998). These dynamic processes force the platesto curl and roll, giving rise to needle-shaped crystals. Needle-shaped crystals are consid-ered ideal for microtomy. Thus the proportion of plates and microcrystals depends on theproportion of high-melting-point and low-melting-point hydrocarbons in the paraffin. Theshape and the size of crystals are influenced by the nature of cell and tissue structures asthe molten paraffin infiltrates and solidifies in the tissue spaces.

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Two types of forces are exerted during paraffin sectioning: flow shearing and point-to-point shearing (Allison, 1998). Flow shearing proceeds ahead of the cutting edge,resulting in smooth sections. In contrast, point-to-point shearing travels through the pathof least resistance ahead of the cutting edge, producing a section of uneven thickness.Paraffin contains additives that minimize the point-to-point shearing and reduce theplastic flow. Additives are synthetic polymers that improve the consistency of paraffin byfilling the spaces among paraffin crystals in the tissue.

Cut a paraffin block containing one tissue specimen with a razor blade,and mount it to a support stub. Trim the block manually with a razor blade or on an auto-matic trimming microtome to a rectangular or trapezoidal cutting face. The size of theblock face is determined by the objective of the study and the size of the tissue specimen.The upper and lower edges of the block facing the knife cutting edge should be parallel toeach other to obtain a ribbon, if required. Mount and orient the block on the microtome,so that its longer edge is parallel to the cutting edge. A steel or glass knife can be used.

Cut sections ( thick) on a rotary microtome, which usually has an automaticadvance mechanism that can be set to advance the specimen block the desired distancetoward the knife with each stroke. Manual or motorized rotary microtomes are commer-cially available (Triangle Biomedical Sciences, Durham, NC; Sakura Finetek, Torrance,CA). A microtome with automated specimen approach, trimming, and sectioning is alsoavailable (Leica Microsystems, Deerfield, IL). Float the sections on water or 4% formalinon a glass slide and heat for 10–15 min at ~40°C on a warming plate to remove the com-pression. Remove the liquid with a fine pipette and dry overnight in an oven at ~40°C toensure section adherence to the slide. Drying can also be accomplished in 15 min at~40°C in a microwave oven. Note: Sectioning will be adversely affected if tissue infiltra-tion with paraffin is incomplete. A too-shallow or too-steep bevel angle of the steel kniferelative to the tissue block face will result in section compression and chatter, respectively.The optimal cutting angle is 4°. Section adhesion to the glass slide can be ensuredby coating the slide with polylysine ( to 1 mg/ml) in 10 mM Tris (pH 8.0).Alternatively, coated slides are commercially available (Probe-On-Plus slides from FisherScientific).

Make sure that the tissue specimen is firmly mounted to the stub and the latter to themicrotome. Also, the paraffin block should be fairly cold at the time of sectioning. Lowhumidity in the vicinity of the microtome tends to result in static electricity, which makesit difficult to separate the section from the block face after cutting. For additional details,see Ruzin (1999).

Silanting of Glass Slides

Detachment of tissue sections from glass slides during processing for immunohistol-ogy is not uncommon. Such detachment can be avoided by preparing silanted slides as fol-lows (Miksys, 1999).

1.2.3.4.

Wash slides in common household detergent.Rinse in running tap water for 10–15 min.Rinse in distilled water.Rinse in 100% acetone for 5 min.

Fixation and Embedding 69

5.

6.7.8.9.

Coat with 2% Aplex (3-aminopropyltriethoxysilane) (Sigma) in 100% acetone for5 min.Rinse in lightly warm running tap water for 2 min.Rinse in distilled water.Dry at ~40°C in a dust-free area.Store at room temperature up to 1 month or at –20°C for several months. Freshlycoated slides are preferred.

Vacuum-Assisted Microwave Heating

A vacuum–microwave combination has been used for processing tissues for lightmicroscopy (Kok and Boon, 1996), transmission electron microscopy of animal tissues(Giberson, 2001) and botanical specimens (Russin and Trivett, 2001), and scanning elec-tron microscopy of human lymphocytes (Demaree, 2001).

The vacuum-microwave heating method is especially useful for processing botanicaltissues because these specimens possess physical characteristics that hamper easy pene-tration of reagents; these characteristics include cell wall, vacuoles, plastids, and intercel-lular spaces. Secondary cell walls may contain cutin, suberin, and lignin, which arehydrophobic. These waxy substances limit the evaporation of water from the tissue andresist the penetration of reagents. The presence of air in the intercellular spaces creates abarrier to fixative penetration. These impediments to reagent penetration and action of fix-atives can be significantly reduced by using vacuum–microwave heating. For details of thismethodology, see Russin and Trivett (2001).

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Chapter 4

Factors Affecting AntigenRetrieval

Many factors influence antigen retrieval, including fixation, heating, retrieval fluid, andantibodies.

FIXATION

Fixation is the most important factor affecting antigen retrieval. Type of fixative andduration and temperature of fixation are all important. Many varieties of epitopes havebeen retrieved with various degrees of success in tissues fixed with formalin, methanol,methacarn, or Bouin’s fixative; buffered 10% formalin containing 3.7–4.0% formaldehydeis the most commonly used. Although fixation with paraformaldehyde or glutaraldehydebetter preserves cell morphology because of stronger, and more rapid and more extensiveprotein crosslinking, antigen unmasking becomes difficult.

The use of formalin has become a matter of habit and convenience, especially inpathology laboratories; it is also inexpensive. To improve the preservation of cell mor-phology, it is recommended that a mixture of formalin (or paraformaldehyde) andglutaraldehyde (0.05–0.5%) be tried. Such mixtures are routinely employed for immuno-cytochemical studies with the electron microscope. It is known that some types of antigensare resistant to fixation with low concentrations of glutaraldehyde. Preembedding immu-nocytochemistry by the avidin-biotin method, which avoids fixative effects, has been suc-cessfully applied for identifying peptide or protein antigens in the brain tissue fixed withglutaraldehyde (Mrini et al., 1995).

The effect of fixation on antigenicity is complex. With the exception of a minority ofantigen types (e.g., PCNA nuclear protein) that are formalin resistant to various degrees,most antigens are sensitive to the concentration of the fixative and the duration of fixation.Although 10% formalin is the usual fixative, it is inadequate for preserving some types ofantigens that are fixative resistant. It has been demonstrated histochemically, for example,that PCNA nuclear protein antigenicity is preserved much better with 20%formalin-PBS than with 10% of the same fixative (Muñoz de Toro de Luque and Luque,1995). The reason is that the protein antigen is partially extracted with the lower fixative

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concentration. In this respect, it should be noted that some types of antigenicity, such asPCNA, require quantitative measurements for assessment of clinical significance, as itslow levels may be present in quiescent cells.

Is antigenicity affected by factors other than fixation? Yes. Although fixation is the mostimportant factor in tissue processing for immunohistochemistry, other factors tend to affectimmunorecognition of antigens. These factors include the interval between removal of thetissue from the human or animal and fixation; the method of excising the tissue from thebody (mechanical damage); the technique of cutting sections of the tissue embedded inparaffin or resin; the procedures for removing section compression, folds, or bubbles andattaching it to the glass slide; the interval between cutting sections and immunostaining (stor-age or without storage of slides prior to staining); and other immunostaining details. Foldsand bubbles hinder section adhesion to the slide, and they may also show 3,3-diaminoben-zidinetetrachloride (DAB) precipitation. Bubbles under sections may appear as brown spotson immunostained sections (Grizzle et al., 2001). Paraffin sections ( thick) adhere tena-ciously to glass slides by heating overnight at 65°C. The use of a PAP pen or other means todemarcate the tissue to aid in staining is also a variable. Awareness of the above-mentionedvariables should prevent erroneously attributing them to problems with fixation.

Tissue specimens ideally should be placed in the fixative immediately after theirremoval from the body. This problem arises in studies of human tissues, for their immedi-ate fixation is usually not feasible. If immediate fixation is not possible, the tissue must bekept cool and moist by covering it with a piece of cloth soaked in sterile, cold saline fornot more than 20–30 min. During this time the specimen should not contact any dry andabsorbent object such as paper, a paper towel, or gauze. To keep the paper trail of the spec-imen (source, time, place of collection, etc.) is no less important.

Note that even human tissues fixed immediately after their removal from the bodymay undergo cellular changes because usually the vascular supply is terminated before thetissue is surgically removed. During this duration (~1 hr) the tissue remains at body tem-perature, at which the activity of digestive enzymes continues, damaging the cellular struc-tures (Grizzle et al., 2001).

Chemical fixation is not the only factor that causes loss or irreversible masking of anti-gens. Treatments such as dehydration and embedding following fixation also play a role inthe loss of immunorecognition of antigens. Absolute ethanol and xylene must not containtraces of water, and the water bath should be very free from contaminants such as bacteria,fungi, dust, and dirt. Once the sections are contaminated, they cannot be decontaminated.

According to Watanabe et al. (1996), the antigen preservation test (Riederer, 1989)showed that immunostaining intensity, for example of decreased during fix-ation with paraformaldehyde but did not decrease during washing and immunostaining.The proportionate decrease in intensity due to fixation was almost constant even when theamount of the antigen differed in the sections. They concluded that the decrease inimmunostaining intensity was related to a proportional decrease in antibody binding dueto masking of antigens during fixation.

DENATURATION

On the basis of antigen retrieval obtained with protein denaturing agents, it has beenproposed that in certain cases antibodies recognize denatured but not native antigens.

Factors Affecting Antigen Retrieval 73

However, this proposition seems untenable, given the requirement of a specific amino acidsequence for an epitope, as well as a specific conformation of antibody molecule, in orderfor antigen-antibody binding to occur. How could an antibody react with a completelydenatured antigen, when the former is usually generated using the native form of thelatter? For example, antibodies generated against selected regions (N-terminal fragment orC-terminal region) of corresponding antigens recognize predominantly similar undena-tured regions, unless these regions of the antigen are masked by other regions of theantigen and/or by some surrounding components.

The proposition that antibodies recognize certain antigens only after the latter havebeen denatured is true only when the epitope is unmasked and remains undenatured fol-lowing antigen denaturation. In other words, a denatured epitope cannot be recognized bythe antibody if the amino acid composition of the epitope peptide and/or its linear aminoacid sequence is altered or damaged. The reaction between the antigen and the antibody isdependent on the conformation of the former. However, the presence of an intact three-dimensional folded antigen structure may not be necessary in certain cases for antibodybinding. Denaturation or unfolding of certain antigen molecules may be necessary tounmask the epitope that is buried in the interior of the folded antigen structure (personalcommunication, Dennis Brown).

It is likely that most antigen molecules form multiprotein complexes, resulting inmasking of epitopes by surrounding proteins. The epitope masking becomes more seriouswhen these complexes are crosslinked with formaldehyde. Such masked epitopes can berecognized by the antibody only when exposed by breaking crosslinks and denaturingsurrounding cell components. If this is so, denaturing treatments cause breakdown ofreversible crosslinks introduced by formaldehyde and denaturation of surrounding cellcomponents, enabling the antibodies to recognize the native, uncrosslinked or partiallycrosslinked undenatured structure of the reactive epitope molecule. It means that denatur-ing treatments do not denature antigen per se but denature multiprotein complexes ofwhich the antigen is a part. It may be that imprecise nomenclature has given rise to con-fusion in this field.

It should also be noted that denaturing agents make cells permeable, facilitating anti-body penetration. Moreover, these agents are used usually in combination with microwaveheating. Therefore, the role of these agents in epitope retrieval needs to be explained inthe context of their role in cell permeabilization as well as of the influence of elevatedtemperatures.

HEATING

Different heating methods, including microwaving, autoclaving, pressure cooking,microwaving combined with pressure cooking, steam heating, and water bath heating,have been employed for antigen retrieval with various degrees of success. However,microwave heating, introduced by Shi et al. (1991), is most commonly used for retrievinga wide range of masked antigens in formalin-fixed and paraffin-embedded tissues.

Heating at a high temperature (100°C) for a short duration (10 min) gives better resultsthan those achieved with a comparatively low temperature for a longer time. However, thereare some exceptions. According to Evers and Uylings (1994a), the immunostaining of SMI-32 obtained at 90°C was superior to that achieved at full power heating.

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pH

Another important factor in achieving optimal antigen retrieval is the pH of the retrievalsolution. It is thought that the pH is more important than the constituents of the retrieval fluid(Shi et al., 1995a). There is, however, no universally optimal pH for a retrieval fluid. Theretrieval of most types of antigens requires a specific pH, although retrieval of a few anti-gens can be achieved over a wide range of pH levels; for example, AE1 and NSE (cyto-plasmic antigens), PCNA (nuclear antigen), and L26 and EMA (cell surface antigens) canbe retrieved at pH levels of 1.0–10.0 (Shi et al., 1995a). On the other hand, followingretrieval at pH 3–6, some antigens (e.g., estrogen receptor) display a marked decreasein the immunostaining, while still other antigens such as cytoplasmic HMB 45 show weakor negative staining after retrieval at pH 1–2 but excellent results in the high pH range(Shi et al., 1995a).

Some antigens can be retrieved only at a low pH. These types are exemplified bythrombospondin and SMI-32 (neurofilament protein), which require pH levels of 1–2 and2.5, respectively (Grossfeld et al., 1996; Evers and Uylings, 1994a). However, pH levelslower than 3.0 can severely damage tissue morphology. Low pH levels can also alter thelocalization of some cytoplasmic antigens, resulting in false-positive staining of thenucleus. For example, an antibody (UCHL1) to T cell antigen is effective at pH 6.0 butresults in the staining of every nucleus at pH 2.0 (personal communication, H.Y. Lan).

In summary, the use of sodium citrate buffer at pH 6.0 increases the intensity andextent of immunostaining of a wide variety of tissue antigens, whereas Tris-HCl buffermay yield better results for some antigens at pH 10.0. On the other hand, low-pH antigenretrieval fluids are necessary for some antigens such as thrombospondin. For previouslyunexamined antigens, a test battery based on three pH values (low, middle, and high)should be carried out to establish an optimal protocol (Shi et al., 1996a).

MOLARITY

The concentration of antigen retrieval fluid is often less important than temperature,duration of heating, and pH in achieving optimal antigen retrieval. For example, sodiumcitrate buffer is effective at molarities ranging from 0.01 to 0.5. However, in the case ofanother antigen retrieval solution, ammonium chloride, 0.5%, 1%, 2%, and 4% solutionswere tested, and 4% concentration yielded the best immunostaining of vimentin inarchival paraffin sections (Suurmeijer and Boon, 1993a). Ammonium chloride solutionsare weakly acidic (pH 3–4). According to Bruno et al. (1992) and Muñoz de Toro deLuque and Luque (1995) minor changes in ionic strength affect the PCNA nuclear proteinantigenicity involved in DNA synthesis. If enzyme digestion methods are used, the opti-mal concentration of the enzyme (e.g., protease) must be applied. Unlike sodium citratebuffer, enzyme solutions cannot be used at a range of concentrations. Note that theconcentration of the diluent used for primary monoclonal antibodies does affect the speci-ficity and intensity of immunostaining (see page 82). It should be noted that the concen-tration of the antibody also affects the specificity and intensity of immunostaining(see page 80).

Factors Affecting Antigen Retrieval 75

ANTIGEN RETRIEVAL FLUIDS

Several antigen retrieval fluids are in use, all having been reported to efficiently medi-ate antigen retrieval. A fluid applicable to all antigens is not available. The main reason isthe enormous variety of chemical structure of not only antigens but also epitopes of anyone antigen. In fact, the chemical nature of the epitope plays a key role in the effectivenessof an antigen retrieval fluid. In other words, the tissue, cell, and antigen types determinethe retrieval fluid. This is substantiated by the fact that generally each type of epitopedetermines the fluid type conducive to its maximal retrieval. A few examples follow.

Two antigen retrieval fluids, sodium citrate buffer (0.1 M, pH 6.0) and glycine-HClbuffer (0.05 M, pH 3.5) containing 0.01% EDTA, were compared for their effectiveness inunmasking a wide variety of antigens (Imam et al., 1995). Glycine-HCl buffer-EDTAyielded stronger immunostaining of p53, androgen, estrogen, progesterone, and Ki-67,whereas sodium citrate buffer produced superior immunostaining of vimentin and leuko-cyte antigens. PCNA was unmasked equally well with either of the two antigen retrievalbuffers, while the two buffers were ineffective in retrieving antigens such as prostatic acidphosphatase and pan-keratin. According to another study, compared with sodium citratebuffer, Tris-HCl buffer (pH 9.5) containing 5% urea yielded more intense staining ofKi-67 in mouse lung tumors (Ito et al., 1998). However, low background staining is likelywhen using the latter antigen retrieval buffer. Certain other types of antigens require acombination of antigen retrieval fluids or systems for their optimal retrieval. Methodsusing such combinations are given in this volume. Comparative effects of antigen retrievalsystems on antigens are summarized in Chapter 6.

In addition to the chemical structure of antigens, a number of other factors, includingpH, heating temperature, molarity, and the chemical composition of the retrieval fluid, areconsidered for selecting the optimal retrieval fluid. Optimal immunostaining of a given anti-gen requires an antigen retrieval fluid of a specific pH. Note that optimal antigen retrievalrequires an optimal fixation procedure. Although the exact mode of action of antigen retrievalfluids is not known, their salts may modify the hydrophobicity of polypeptide chains, affect-ing the conformation of protein molecules. The major effect of the salts is to mediate hightemperature effects. However, this mechanism does not explain the mode of action of non-buffer fluids (e.g., water) used for antigen retrieval. Excellent p53 immunostaining in breasttumors has been achieved by heating the sections in water for 15 min at 50°C (Katoh andBreier, 1994). Heating is carried out in a microwave oven or in a water bath.

Antigen retrieval can occur under both acidic and alkaline conditions, depending onthe type of antigen involved. The mechanisms involved in the antigen retrieval at differentpH values are not known. Three commonly used antigen retrieval fluids are 0.01 M sodiumcitrate buffer (pH 6.0), 0.01 M Tris-HCl buffer (pH 1.0) or 0.1 M Tris-HCl (pH 10.0), and0.05 M glycine-HCl buffer (pH 3.6). The latter can be used with or without 0.01% EDTAdepending upon the antigen type. Taylor et al. (1996b) recommend 0.1 M Tris-HCl buffer(pH 9.5) containing 5% urea. These fluids provide strongly alkaline or acid environmentsand are effective for antigen retrieval in tissues which have been either mildly fixed oroverfixed with formalin. These recommendations are based on the successful immunos-taining of a wide variety of antigen-antibody complexes. For most clinical applications,0.01 M sodium citrate buffer (pH 6.0) is recommended.

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Only if sodium citrate buffer or Tris-HCl buffer fail to yield satisfactory retrievalshould fluids containing substances such as EDTA, EGTA, enzymes, metal salts, periodicacid, or urea be tried. Consider the immunostaining of parvalbumin, calbindin, and MAP,which has been found to be best accomplished using 4% aluminum chloride (Evers andUylings, 1994b). However, in this study the antigen retrieval effect of the metal solutionwas compared only with that of distilled water and zinc sulfate; sodium citrate buffer wasnot tested. Since free-floating vibratome sections of the human brain tissue underwentsevere wrinkling during microwave heating, thick tissue slices (0.5 cm) were placed in 4%aluminum chloride solution and heated in a microwave oven for 10 min, followed by stan-dard immunocytochemical staining of semithin sections Another advantage ofpretreatment is that the brain tissue hardens, facilitating easier sectioning.

Fluids other than standard sodium citrate and Tris-HCl buffers are also preferred insome other cases. An example is the retrieval of Bcl-2 antigen (oncoprotein), which is bestachieved by hydrated autoclaving of sections placed in deionized water (Umemura et al.,1995). Immunostaining of neurofilament proteins, proliferating cell nucleus antigen(PCNA), retinal S-antigen, and glial fibrillary acidic protein (GFAP) has been obtained byusing distilled water as the antigen retrieval fluid in a microwave oven (Yachnis andTrojanowski, 1994). However, heating in water in a microwave oven is not generally rec-ommended. Neurofilament proteins in archival tissues have been immunostained afteremploying a saturated solution of lead thiocyanate (Yachnis and Trojanowski, 1994). Zincsulfate has been used for retrieving vimentin and prostate-specific antigen (Wieczoreket al., 1997). Cesium chloride (5.7 M) has also been employed for antigen retrieval.However, such metal salt solutions are not recommended because they are toxic. Targetunmasking fluid (TUF) was developed by van den Berg et al. (1993) for routine immuno-histochemistry and is commercially available (Signet Lab, Delham, MA, or KreatechBiotechnology, Amsterdam).

Periodic acid (0.5%) has also been used as an antigen retrieval fluid (Xue et al., 1998).Another type of antigen retrieval fluid is EDTA of pH 8.0 (Morgan et al., 1994; Pileri et al.,1997), which has been stated to be effective irrespective of the location of the target mole-cule (intranuclear, intracytoplasmic, or membrane-bound). Comparative studies by Eharaet al. (1996) also indicate that EDTA (0.15 M, pH 6.0) yields stronger immunostaining ofsteroid hormone receptors than that obtained with sodium citrate buffer (0.01 M, pH 6.0).However, the preservation of cell morphology is superior when citrate buffer is used. Urea(3 M), formic acid, and guanidine solutions have also been employed for antigen retrieval.When urea is used in an autoclave or a pressure cooker, it has the disadvantage of yieldingfalse-negative results or background staining (Shi et al., 1996b). In another study, a satu-rated solution of dimedone was applied for antigen retrieval (Shi et al., 1996b). Recently, itwas reported that the addition of calcium chloride to the antigen retrieval fluid of a low pHimproved the preservation of tissue morphology (Morgan et al., 1997a,b).

Boric acid (0.2 M, pH 7.0) in conjunction with low-temperature, heat-mediated anti-gen retrieval technique has been successfully used as the antigen retrieval fluid for estro-gen receptors on freshly cut sections of breast tissue (Peston and Shousha, 1998). Boricacid is also very effective in antigen retrieval on the archival hematoxylin-eosin-stainedlymphoid sections on coated or uncoated slides, using conventional heat-mediated antigenretrieval method (Biddolph and Jones, 1999). Lymphoid sections tend to dislodge from thecoated or uncoated slides in the presence of sodium citrate buffer during antigen retrieval.

Factors Affecting Antigen Retrieval 77

The above discussion indicates that although 0.01 M sodium citrate buffer (pH 6.0) iscommonly used, it is not a universally ideal antigen retrieval fluid for all types of tissuesand antigens. If published information is not available with regard to the best antigenretrieval fluid for the antigen under study, the ideal retrieval fluid for each type of epitopemust be determined by trial and error.

The following four antigen retrieval fluids are commercially available (BioGenex,San Ramon, CA). The approximate pH indicated below is valid at the time of manufac-ture; the pH of the fluid may change during storage.

1.2.3.4.

Antigen Retrieval Citra Microwave Solution used at pH 6.0.Antigen Retrieval Citra Plus Microwave Solution used at pH 6.1.Antigen Retrieval Glyca Microwave Solution used at pH 3.5.Antigen Retrieval AR-10 Microwave Solution used at pH 10.5.

Other commercial sources for antigen retrieval fluids are:

1.

2.

Dako TRS, Dako Corporation, 6392 Via Real, Carpinteria, CA 93013 HIERbuffer, Ventana Medical Systems, Tucson, AZTarget Unmasking Fluid (TUF*), Monosan (Sanbio), Fronstraat 2A, Postbus 540,AM Uden, NL-5402, The Netherlands; Serotec Ltd., 22 Bankside, StationApproach, Kindlington, Oxford, Oxon OX5 1JE, U.K.

Glycerin as Antigen Retrieval Fluid

When other antigen retrieval methods fail, antigen retrieval can be accomplished in90% glycerin solution using a hot plate with a magnetic stir rod. This approach is thoughtto improve preservation of tissue morphology as well as efficient retrieval of some anti-gens. Glycerin has the advantage of having a very high boiling point (290°C) and beingnontoxic, stable, and reusable. The stir bar maintains a constant and uniform temperaturethroughout the antigen retrieval fluid, prevents hot or cold spots, and thus facilitates reli-able and consistent results. This method can also be used in a conventional hot air oven.Many slides in metal slide racks can be processed simultaneously in this oven. Glycerinsolution can also be used for antigen retrieval in Coplin jars in a microwave oven; an emptyspace must be kept between the slides.

The glycerin method has been used for retrieving a number of antigen types, includ-ing estrogen receptor (Beebe, 1999). It is especially useful for very small, fragile biopsiessuch as prostate needle biopsies and bowel biopsies. Pure glycerin fails to bring about anti-gen retrieval, which means that water and heat are required to cleave the formaldehydemolecule from the proteins, break down the methylene bridges, or rehydrate the proteins.The exact role of glycerin in the antigen retrieval mechanism is not known. Further testingof the usefulness of this procedure is awaited.

*Contents: chromium potassium sulfate dodecahydrate, sodium dodecyl sulfate, dextran sulfate, formamide,phosphate salt, magnesium sulfate, pepsin, polyethylene glycol, and Triton. It has a low toxicity and is irritat-ing to the eyes and skin. It is a colorless, nonviscous liquid.

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Procedure

The antigen retrieval fluid consists of a mixture of 100 ml of 90% glycerin and 10 mlof Dako’s citrate buffer. The slide rack is placed in a bowl of an appropriate size and shape,so that the stir bar rotates freely under the slide rack. A sufficient volume of antigenretrieval fluid is transferred to the bowl ~ 1 cm above the level of the slides. The hot plateis turned on and adjusted until the temperature of the fluid reaches 100–120°C; the dura-tion of heating varies between 5 and 20 min. For estrogen and progesterone retrieval theduration of heating (120°C) is ~7 min with a 15-min cool-down period before the slidesare transferred to distilled water for further processing.

Alternatively, such high temperatures in the bowl can be achieved by placing it in amicrowave oven, then removing and placing it on the hot plate. This is followed by addingthe stir bar and the slide rack to the bowl. It takes 1 min for 100 ml of the glycerin solu-tion to attain a temperature of 125°C in a 600 W microwave oven on high. If more than100 ml of solution is to be heated to the same temperature, for every additional 100 ml anadditional 1 min is required.

pH of Antigen Retrieval Fluids

In addition to heating, retrieval fluid pH plays a key role in achieving optimal antigenretrieval. It is thought that the pH is more important than the composition of the retrievalfluid. This is supported by the demonstration that optimal staining of antigen SMI-32 wasachieved at pH 2.5 and 2-hr microwave heating at 90°C, whereas staining of antigen MAP-2 was best obtained at pH 4.5 and 10-min full-power heating; in both cases 0.05 M citratebuffer was used (Evers and Uylings, 1994a). Therefore, in optimizing the antigen retrievalprotocol, pH is a priority.

Note that there is no universally optimal pH for a retrieval fluid. The retrieval of eachtype of antigen requires a specific fluid pH, although exceptions occur with antigens thatcan be retrieved at a wide range of pH levels. For example, AEI and NSE (cytoplasmicantigens), PCNA (nuclear antigen), and L26 and EMA (cell surface antigens), can beretrieved at pH levels of 1.0–10.0 (Shi et al., 1995a). On the other hand, some antigens(e.g., estrogen receptor) display a marked decrease in immunostaining at pH 3–6, whilestill other antigens, such as cytoplasmic HMB45, show weak or negative staining atpH 1-2 but excellent results in the high pH range (Shi et al., 1995a).

Some antigens are retrieved only at a low pH. These types are exemplified by throm-bospondin and SMI-32 (neurofilament protein), which require pH levels of 1–2 and 2.5,respectively (Grossfeld et al., 1996; Evers and Uylings, 1994a). Note, however, that pHlevels lower than 3.0 can severely damage tissue morphology, especially with intense heat-ing. Low pH levels can also alter the localization of some cytoplasmic antigens, resultingin false-positive staining of the nucleus. For example, an antibody (UCHL1) to T cell anti-gen is effective at pH 6.0 but results in the staining of every nucleus at pH 2.0 (personalcommunication, H. Y. Lan).

Generally Tris-HCl buffer produces better results at higher pH levels (e.g., pH 10.0)than do some other buffers. On the other hand, sodium citrate buffer increases the intensityand extent of immunostaining of a wide variety of tissue antigens at pH 6.0. EDTA-NaOH(1 mM) at pH 8.0 also yields satisfying results. Although relatively high pH solutions, such

Factors Affecting Antigen Retrieval 79

as sodium citrate or Tris-HCl, are suitable for most antigens, low pH solutions are pre-ferred for nuclear antigens (Taylor et al., 1996a). However, solutions of low pH generallytend to cause weak focal background staining and damage to some epitopes. A test batterybased on three pH values (low, middle, and high) should be carried out to establish anoptimal protocol, including pH, for immunostaining previously unexamined antigens(page 104).

Ionic Strength of Antigen Retrieval Fluids

The ionic strength of the fluid in which tissues are suspended during fixation withformaldehyde, unlike retrieval fluid concentration, does influence antibody access to intracel-lular antigens such as proliferation cell nuclear antigen (PCNA), nuclear protein (Ki-67)detected by MIB-1 antibody, and nuclear antigen p120. Ionic bonds are known to be respon-sible for a major portion of protein-protein interactions, and their breakage causes dissocia-tion of the interacting proteins, resulting in increased detectability of the antigen. Suchbreakage occurs with increased salt (NaCl) concentrations. It has been shown that theimmunofluorescence of antigens such as PCNA is increased when the cells are fixed in thepresence of increased salt concentrations (Bruno et al., 1992). The increase is greater for cellsin the phase of the cell cycle than for cells in S or phase. High salt concentrations loosenthe proteins, which are then stabilized with formaldehyde. In other words, increased ionicstrength weakens intra- and intermolecular ionic interactions during the process of crosslink-ing with formaldehyde. Using the optimal ionic strength of the solution, which must be cus-tomized for a given antigen, will facilitate the accessibility of the antibody to the epitope.

ANTIBODY PENETRATION

The fundamental question in the phenomenon of antigen retrieval is whether it is dueto enhanced penetration of antibodies into the tissue or to reversal of protein conforma-tional changes induced by fixation, or both. The evidence favors both explanations. All thetreatments (microwave, autoclave, and conventional heating, enzyme digestion, ultrasoundapplication, and detergent treatment) used for antigen retrieval break down proteincrosslinkages, facilitating antibody access to the antigen. The achievement of increasedimmunostaining after using cell permeabilization methods testifies to the role of antibodypenetration into the tissue. The above treatments also restore the original conformationof the protein molecule, resulting in enhanced interaction between the antigen and theantibody.

Antibody molecules are relatively large. The speed and extent of antibody penetrationinto the tissue and the degree of fixation with formaldehyde are inversely related. In otherwords, the stronger the fixation (protein crosslinking), the slower the antibodypenetration. The reason for this relationship is that extensive crosslinking results in the for-mation of a compact protein network which impedes antibody penetration. Such an imped-iment can be caused by the cell membranes as well as by the cytoplasmic matrix, both ofwhich contain proteins. It is therefore apparent that strong, extensive protein crosslinkingshould be avoided before incubation with antibodies. This is the reason for preferring

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formaldehyde over glutaraldehyde, since the latter forms strong protein crosslinkages. It isalso well established that tissue specimens that have been fixed for a long time (weeks ormonths) require more vigorous treatments of sections for antibodies to penetrate and haveaccess to the antigens. It is interesting to note that an antibody against a specific epitopeless sensitive to aldehyde fixation can be obtained by immunizing the mice with an anti-gen in which the aldehyde-sensitive epitope has been blocked or altered.

To increase the penetration of antibodies into thin resin sections of fixed tissues, simul-taneous heating of sections and antibodies has been attempted. This treatment is thought toincrease the labeling of certain antigens, whereas that of some other antigens remains unaf-fected. It has been demonstrated that such a treatment enhanced labeling density by the anti-amylase antibodies, whereas labeling with anti-DAMP antibodies remained unchanged(Chicoine and Webster, 1998). Further developments of this protocol are awaited.

ANTIBODY DILUTION

Not only the type (e.g., the cell clone) and the source of availability of an antibodybut also its dilution are important in fully utilizing the effectiveness of an antibody as apowerful tool to detect antigens. The optimal antibody concentration for antigen varies,depending on whether the tissue used is aldehyde-fixed or frozen; generally higher anti-body concentrations are required for sections of aldehyde-fixed tissues (Fig. 4.1).

Also, different forms of an antigen require different concentrations of the antibody fortheir maximal detection. This is exemplified by the PC-10 primary antibody, which identi-fies PCNA antigen at a dilution of 1:1000 in epithelial cells in normal colon tissue, whereasa dilution of 1:400 is required to localize these proliferating cells in adenomatous polyps(Holt et al., 1997). In contrast, some types of antigens (e.g., Ki-67) can be optimally detectedin various tissue types at the MIB-1 dilution of 1:50, using the microwave heating antigenretrieval method. However, in a few studies MIB-1 dilutions of 1:20 to 1:100 have been used.

There are many pitfalls, including false-positive and false-negative staining, to usingantibody concentrations of higher or lower than optimal concentrations. Labeling speci-ficity partly depends on the antibody dilution, while background staining criticallydepends on its dilution. Also, the esthetic appeal of the images produced by immunohis-tochemistry diminishes with suboptimal dilutions of antibodies. Therefore, to avoidunwanted staining, pay careful attention to the optimal working dilution of an antibody,especially of a polyclonal antibody, and to washing procedures. Also note that antigenretrieval treatments allow the use of increased dilutions of the antibodies. For example, forsections of formalin-fixed and paraffin-embedded tissues, the optimal dilution of PC-10antibody without heat pretreatment is 1:10 compared with 1:600 after microwave heating(Haerslev and Jacobsen, 1994).

A fairly high concentration of the primary antibody is necessary to follow saturationkinetics. However, the majority of these antibodies exhibits a bell-shaped concentration-binding curve, with the binding increasing up to a specific antibody concentration and thendecreasing. Such a bell-shaped curve is due to unstable binding of the antibody to the anti-gen under very high antibody concentrations. Effects of a high antibody concentration canbe examined with the method of Raivich et al. (1993). In practice, however, one rarely

Factors Affecting Antigen Retrieval 81

aims for or achieves saturation kinetics in routine immunohistochemistry. In most cases weaim for and achieve adequate and reproducible staining.

High concentrations of primary antibodies can increase nonspecific binding and alsocompromise antigen-specific immunostaining. Furthermore, antibodies may aggregate athigh concentrations, which limits their penetration. Electrical charges on aggregated anti-bodies may hinder their penetration among similarly charged cell molecules; therefore,antibodies should be used at concentrations at or slightly below antigen-specific concen-trations. Note that similar antibodies obtained from different sources may not yield thesame intensities of immunostaining.

Some evidence indicates increased labeling efficiency of certain diluted antibodies(e.g., antiamylase antibodies and anti-MHC class II antibodies) when they are exposed tomicrowave heating prior to their application (Chicoine and Webster, 1998). However, such

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increased labeling may be accompanied by enhanced background staining. Furthermore,such a treatment reduces labeling density of some other antibodies such as anti-DNP anti-bodies. The duration of heating of antibodies is critical to obtain optimal labeling. Theoptimal heating duration varies with the antibody and the fixation parameters used to sta-bilize the cellular components. The mechanism(s) responsible for increased or decreasedlabeling efficiency of different antibodies as a result of their heating is not known.

Diluent Buffer for Primary Antibodies

Both the pH and osmolarity of the diluent buffer (especially the type of solute) affectthe affinity of monoclonal antibodies for the antigen. It is known that electrolytes exert aprofound effect, not only on the structural relations of protein molecules, but also on thereactivity of proteins (Hayat, 2000a). The reactivity of both monoclonal and polyclonalantibodies with antigens is affected by the type of antibody diluent used. This is truewhether or not a heat-induced antigen retrieval is used. Optimal pH increases the sensitiv-ity (staining intensity) as well as the specificity of immunostaining. Acceptable shelf-lifeof antibodies can also be achieved at optimal pH and dilution in the presence of stabiliz-ing protein (Boenisch, 1999). Unfavorable pH diminishes immunoreactivity because itreduces antibody affinity for the antigen. The role of pH in the interaction between theantibody and the antigen in immunohistological processing is explained below.

Antibodies are attracted to the epitopes of most glycoproteins and polypeptides ini-tially through electrostatic charges and subsequently through van der Waals andhydrophobic interactions (Boenisch, 1999). In immune reactions, the isoelectric point (pI)of both antigens and antibodies is therefore of importance. The pI of polyclonal IgGsranges from 6.0–9.5. Monoclonal antibodies of at least this class possess an equallywide range of individual pI values. If the pH of the diluent and/or solute is used in the samerange, the result will be changes in both electrostatic charge and conformation of at leastsome monoclonal antibodies and possibly of some reactive epitopes. Antibody configura-tion controls spatial complementarity. All these changes contribute to variable attractionbetween the antibody and the antigen.

As stated above, the pH of the diluent affects the electrostatic charge of monoclonalantibodies and thus the interactions between the antibody and the reactive epitope.Consequently, the optimal operational pH of the monoclonal antibody is determined by theelectrostatic charge of the paratope and that of the epitope. Most effective initial attractionbetween the paratope and the epitope occurs at the pI intermediate to the antigen and thatof the antibody. For most antibodies, but not all, this pH is mildly acidic (6.0). An increas-ingly higher pH of the diluent will decrease the net positive charge of most monoclonal anti-bodies, resulting in their reduced attraction to negatively charged target epitopes. Higher pHvalues will also increase the hydrophobicity of antibodies, lessening the interaction betweenthe antigen and the antibody because of the decreased penetration by the latter.

It is suggested that new monoclonal antibodies be tested in several dilutions higherthan those recommended by the vendor, using 0.05 M Tris buffer (pH 6.0 and 8.6)(Boenisch, 1999). The highest dilution and the pH at which maximal staining occursshould be determined for routine use of the new antibody. This approach frequently allowsfor the use of antibody dilutions much higher than those recommended by the supplier.

Factors Affecting Antigen Retrieval 83

Note that the use of higher concentrations of monoclonal antibodies does not improveweak staining in immunohistochemistry because of paucity of or masked antigens.

Although PBS is commonly used as the antibody diluent, this solution has certain dis-advantages. Sodium ions in PBS tend to shield negatively charged epitopes, thereby dimin-ishing the attraction of positively charged reactive sites on the antibody, especially at analkaline pH. Phosphate ions, on the other hand, promote hydrophobicity. Therefore, theuse of PBS and phosphate buffer for diluting the antibodies is not desirable. Accumulatedevidence indicates that the most suitable diluent for both monoclonal and polyclonal anti-bodies is 0.05–0.1 M Tris buffer (pH 6.0) (Boenisch, 2001). The advantage of this pHbecomes clear when considering that most antigens and monoclonal antibodies possessopposite surface charges at pH 6.0. Thus, to achieve optimal immunoreactivity, the pH ofthe environment should be intermediate between the pH of the antigen and that of themonoclonal antibody. Note that any change in the composition and pH of the diluentaffects the performance of both the antibody and the antigen.

In addition to the pH, the osmolarity of the buffer used to dilute the primary antibodytends to influence the immunoreactivity of monoclonal antibodies. The changes in themolarity of Tris buffer used for diluting monoclonal antibodies are expected to result inchanges in the immunoreactivity of antibodies. It has been reported that the higher the con-centration of cations (e.g., ) in the buffer or the higher the pH in their presence, theless the immunoreactivity of the monoclonal antibodies (Boenisch, 1999). However, poly-clonal antibodies may not show such an adverse effect.

STORAGE OF PARAFFIN-EMBEDDED TISSUES

Antigenicity is preserved much better in paraffin-embedded tissue blocks during stor-age than on the paraffin sections. However, an agreement is lacking regarding the loss ofantigenicity due to storage of formalin-fixed and paraffin-embedded tissues. Even similartissues processed in a similar fashion and stored for the same period of time in differentlaboratories may show differences in the degree of immunoreactivity. Also, the use of thesame antibody when used in different tissues stored for the same duration may yield dif-ferent degrees of immunohistochemical staining. This predicament is exemplified by theandrogen receptor.

Androgen receptor activity has been reported to be preserved in the sections of for-malin-fixed, paraffin-embedded human archival benign prostate tissue that was stored forup to 16 years (Janssen et al., 1994). In this study monoclonal antibody F39.4 was used; itwas raised against a synthetic peptide (SP61) corresponding to the human androgen recep-tor amino acid sequence 301–320 of the N-terminal domain.

In contrast, it has been demonstrated that a significant and persistent decrease in theandrogen receptor immunoreactivity occurred in prostatic adenocarcinomas when theywere stored for 2 years (Dash et al., 1998). The antibody used was F39.4.1 (BioGenex, SanRamon, CA). Such an immunoreactivity decreased to near zero after 4 years. This decreasebegins slowly, followed by more rapid decline, and finally again slows down. In this study,antigen retrieval with microwave heating did not negate the adverse effects of the storageof tissue blocks. Methyl green was used for identifying the tissue background and high-lighting the nuclei. Image acquisition and analysis were performed with a CAS 200 image

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analyzer (Becton Dickenson Cell Analysis Systems, Mountain View, CA). Antigenretrieval in both of these studies was carried out using microwave heating. If the preserva-tion of androgen receptor antigenicity is a problem in a formalin-fixed archived tissue, oneway to circumvent this problem is to use archived fresh-frozen tissues. On the basis of theirstudy, Dash et al. (1998) have suggested that the use of this antibody for retrospective stud-ies does not correlate androgen receptor status with prognosis or therapeutic response. Adecrease in immunoreactivity of p53 antigen in stored paraffin sections is discussed onpage 85.

STORAGE OF TISSUE SLIDES

Sections of formalin-fixed and paraffin-embedded tissues are commonly used todetect antigens of diagnostic, therapeutic, or prognostic importance in patients with manytypes of cancers. Therefore, the need for accuracy in the detection of antigen is obvious.Although sections of these tissues are mounted on glass slides usually 1 or 2 days beforestaining, in some cases paraffin blocks are no longer available. Consequently, immunohis-tochemistry must be performed on unstained slides that have been prepared some timeago and stored. Because antigen alterations occur on unstained, stored paraffin sections,factors responsible for the alterations need to be understood in order to increase the relia-bility and quality of immunohistochemical studies (Hayat, 2000a).

Most of the technical factors that positively or negatively affect antigen detections arediscussed elsewhere in this volume. The following discussion is limited to the clarificationof factors influencing the detection of antigens on the stored paraffin sections. Generally,prolonged storage of sections at room temperature results in decreased immunostaining,and thus false-negative staining, which may lead to diagnostic errors and inaccurate prog-nostic information (Fig. 4.2/Plate 1B-E). In general, antigens that do not require antigenretrieval assistance are less adversely affected during storage than those requiring antigenretrieval with microwave heating or enzyme digestion. Membranous antigens seem to bemore adversely influenced by storage of sections than are those located in the cytosol andthe nucleus. It is emphasized that antigens affected during storage of sections showdecreasing staining with increasing storage temperatures because the stability of most anti-gens remains intact during storage at 4°C. Decreased immunoreactivity caused by sectionstorage can be compensated for in most cases by using the optimal antigen retrievalmethod. Different adhesives, such as gelatin and poly-L-lysine, used to ensure sectionadhesion to the slide do not influence antigen preservation during storage (van den Broekand van de Vijver, 2000).

Admittedly, the effect of storage of paraffin-embedded tissue sections on the extentand intensity of immunostaining is controversial. A number of contradictory reports havebeen published, especially regarding the effects of the type of fixative, duration of fixationwith formalin and other fixatives, and temperature of storage (Leong and Gilham, 1989b;Miettinen, 1989; Malmström et al., 1992; Hendricks and Wilkinson, 1994; Bromley et al.,1994; Prioleau and Schnitt, 1995; Kato et al., 1995; Jacobs et al., 1996; McDermott et al.,1997; Shin et al., 1997; Songun et al., 1997; Bertheau et al., 1998; Grabau et al., 1998;Dwork et al., 1998). Recently, excellent, detailed studies of this problem have been carried

Factors Affecting Antigen Retrieval 85

out by Wester et al. (2000) and van den Broek and van de Vijver (2000). These studies andmy personal experience are reviewed below.

Only a few studies report that prolonged storage of sections does not adversely affectantigen detectability. For example, according to Williams et al. (1997), long-term(6 months at room temperature) storage of sections of tonsil tissue had no effect on thereactivity of the five antibodies tested. In contrast, a vast number of other studies demon-strate decreased staining of stored sections, especially when they are stored at room tem-perature. It has been demonstrated, for example, that antigens such as p53 and Ki-67 (lungand breast carcinoma) show lower staining after storage for 3 years at room temperaturethan sections stored for the same period of time at 4°C or –80°C (Grabau et al., 1998).Nuclear estrogen receptor in breast carcinoma also shows higher reactivity when deparaf-finized sections are stored for up to 4 weeks in 10% sucrose in PBS at 4°C than that shownby sections stored at room temperature for the same duration (Bromley et al., 1994). Thisincreased staining could be the effect of cold temperature and PBS in which the sucrose isdissolved. It is also known that dissolved salt solutions unmask epitopes in formalin-fixedand paraffin-embedded tissues.

It has also been demonstrated that the staining of p53 in mammalian ductal carcinomadecreased after slides were stored for 2 months at room temperature, but the antigen loss

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was significantly less when slides were stored at 4°C (Jacobs et al., 1996). A gradual lossof staining of Ki-67 was reported in the colon tissue when the slides were stored for9–21 days at room temperature before staining, although a delay of 5 days did not dimin-ish the staining (Holt et al., 1997). Maintaining cut sections refrigerated and protectedfrom light failed to prevent such a loss of MIB-1 immunoreactivity with time. On the otherhand, when sections were stored for as long as 1 month at room temperature before stain-ing of PCNA antigen in the same tissue, no adverse effect was observed on the immunore-activity of PC10 antibody with this antigen.

According to Shin et al. (1997), p53 immunoreactivity was not decreased with stor-age of slides for as long as 25–48 months at room temperature, provided staining intensityis not the only objective of the study. The percentage of positivity of microwave-enhancedimmunoreactivity of p53 stored at room temperature and fresh paraffin sections was notstatistically significant. Nevertheless, the staining intensity of heated, stored sections wasstronger than that in nonheated, freshly cut sections. This study was carried out usingtissue blocks of head and neck squamous cell carcinomas stored for 4–15 years and lungcarcinomas stored for 14–25 years. Zinc sulfate (1%) was used as the antigen retrievalfluid and was heated for 3 min in the microwave oven. Similarly, the immunostaining ofp53 antigen in sections of colorectal carcinoma stored for 6–14 months at room tempera-ture was excellent after microwave heating (Kato et al., 1995). The aforementioned dis-agreement is due to the study of p53 antigen in different tissues and/or differences in thedetails of the methodologies used in different laboratories.

There are many reasons for the lack of consensus on this highly complex phenome-non, and they are discussed below. Various studies mentioned above were conducted usingdifferent parameters of antigen retrieval methods, including antigen retrieval fluids, pH,heat source, temperature, and duration of treatments for detecting different antigens. Thetype of fixation and duration of fixation also varied in these studies. Other variants werethe type of epitope and antibody and source of antibodies used.

The degree of immunostaining of stored paraffin sections may differ, depending onwhether monoclonal or polyclonal antibodies are employed. Polyclonal antibodies haveaffinity for several types of epitopes, resulting in positive staining, which may be nonspe-cific. An antigen retrieval method unmasks more than one type of epitope on the samesection, whether stored or not, and such epitopes have access to the polyclonal antibody.However, a recent study indicates that polyclonal antisera show only slightly betterstaining than that obtained with monoclonal antibodies (van den Broek and van de Vijver,2000). It is also possible that loss of immunostaining in stored sections is epitope relatedinstead of related to the antigen as a whole (Henson, 1996).

In addition, tissue heterogeneity at different levels was not considered. The quantityand quality of antigen reactivity varies from one tissue block to another. In some of thesestudies the automated immunostainer was used, whereas in others manual staining wascarried out. The automated stainers reduce human procedural errors, and their controlledenvironment increases the speed and timeliness of results in high-volume laboratories,although their high cost might be prohibitive for small laboratories. Another factor that mayaffect results is that a collection of stored sections may be heterogenous regarding dura-tion of storage because of the successive addition of new sections. Subjective evaluationor inadequate quantification of the extent and intensity of staining was the common

Factors Affecting Antigen Retrieval 87

denominator in most of these studies. These are the main reasons for the contradictoryresults reviewed above.

Attempts have been made to protect sections from exposure to oxygen during storageby coating them with paraffin (Jacobs et al., 1996; Rittman, 2000). This is accomplishedby heating the slide to ~60°C and placing a few drops of molten paraffin on it; a secondheated slide is gently drawn across the surface of the first slide to form a thin protectiveparaffin layer on the sections. In our experience, coating the surface of paraffin sectionsmounted onto slides with paraffin does not significantly reduce antigen loss after their stor-age for several weeks or months at room temperature or at 4°C. Xylene-based spray gluehas also been used for protecting the stored sections but without success (Wester et al.,2000).

Different antigens are affected differently by the storage of sections of even the sameformalin-fixed, paraffin-embedded tissue. In other words, the degree of the antigenicityloss due to storage of the unstained slides differs depending on the type and location of theantigen. For example, nuclear steroid receptors tend to be comparatively more sensitive tostorage (aging). Comparative studies, using a panel of eight antibodies against Ki-67,prostatic-specific antigen, androgen receptor, epidermal growth factor receptor, and pro-static acid phosphatase, demonstrated that nuclear androgen receptor showed a higherdecrease of antigenicity in stored, unstained sections compared with that exhibited byother antigens (Olapade-Olaopa et al., 2001).

The loss of antigens due to storage of paraffin sections is not a serious problem inmany clinical immunohistochemistry laboratories that perform immunostaining withinhours or days after paraffin sections have been cut. However, antigen loss may become aproblem when slides are stored for months at room temperature as positive controls. Suchstorage is encountered in some research laboratories where unstained paraffin sections arearchived for future use. In any case it is recommended that sections be stained rapidly afterthey have been cut from the formalin-fixed, paraffin-embedded tissues. It is likely that pro-longed storage of sections at room temperature strengthens protein crosslinks, whichbecome less reversible, resulting in diminished antigen retrieval. If immunostaining needsto be postponed, tissue specimens should be stored in paraffin blocks rather than as paraf-fin sections because antigenicity is better preserved in the former state. The results of acomprehensive study on the effects of fixation, temperature and duration of sectionstorage, and antigen retrieval on the immunostaining of p53 antigen are shown inFig. 4.3 (Plate 2). Color-based image analysis was used to quantify the extent and inten-sity of staining.

In conclusion, the storage of paraffin sections decreases, to a varying degree,immunoreactivity for most, but not all, antigens. The maximal decrease in immunoreac-tivity, at least of p53 and Ki-67 antigens, occurs during the first 2 weeks of storage (Westeret al., 2000). The decrease in immunoreactivity is generally inversely related to an increasein storage temperature. Both the extent and intensity of staining tend to be negatively influ-enced by storing the sections. The decreased immunoreactivity as a result of section stor-age can be compensated for in most cases by using optimal antigen retrieval procedure.Although longer durations of fixation are accompanied by increased masking of antigensduring section storage, this relationship is neither universal nor linear. If paraffin sectionsmust be stored, they should be stored at –20°C, irrespective of the duration of storage.

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Factors Affecting Antigen Retrieval 89

SIGNAL AMPLIFICATION

Most antigens are not destroyed during fixation with formaldehyde, but are reversiblymasked. Methods to unmask them are presented in this volume. However, some antigensare difficult to visualize adequately with routine immunohistological techniques and there-fore require signal amplification with an acceptable signal-to-noise ratio. An amplificationof immunostaining intensity is especially useful when monoclonal antibodies are usedbecause they bind only a single epitope. Small amounts of antigens in tissue sections canbe detected specifically by using signal amplification. Techniques used for increasing thesensitivity or signal amplification are summarized below.

A number of strategies have been employed for improving immunohistochemical sig-nals. The immunofluorescence antibody method was developed for specific identification ofcells based on their antigen makeup (Coons et al., 1941). Its use is limited because of the needfor fresh-frozen sections and inadequate preservation of cell morphology. Also, the fixed ratioof fluorescein to the antibody does not allow amplification of the signal. The peroxidase-labeled antibody method is more compatible with the basic substrates of surgical pathologyspecimens fixed with formalin and embedded in paraffin (Nakane, 1968). This immunoper-oxidase protocol can be amplified by increasing the duration of development.

The original immunoenzyme bridge method using enzyme-specific antibody (Masonet al., 1969) has been superseded by an improved technique using a soluble peroxidaseantiperoxidase complex (PAP) (Sternberger et al., 1970). These complexes are formedfrom three peroxidase molecules and two antiperoxidase antibodies and are used as a thirdlayer in the staining method. They are bound to the unconjugated primary antibody (e.g.,rabbit antihuman IgG) by a second layer of bridging antibody (e.g., swine antirabbitimmunoglobulin), which is applied in excess so that one of its two identical binding sitesbinds to the primary antibody and the other to the (rabbit) PAP complex. The PAP methodis more sensitive than indirect methods using fluorescein or peroxidase-conjugated antis-era. Alkaline phosphatase antibodies raised in the mouse can, by the same principle, beused to form alkaline phosphatase anti-alkaline phosphatase (APAAP) complexes. Thesehave uses and advantages similar to those of the PAP complexes.

The avidin-biotin methods rely on the marked affinity of the glycoprotein avidin forbiotin. Avidin is composed of four subunits which form a tertiary structure possessing fourbiotin-binding hydrophobic pockets. The oligosaccharide residues in avidin give it someaffinity for the tissue components, especially some lectinlike proteins, and result in non-specific binding. A similar molecule, streptavidin, has some advantages over avidin, as theformer lacks oligosaccharide residues and possesses a neutral isoelectric point.

The low-molecular-weight vitamin biotin is easily conjugated to antibodies andenzyme markers. Up to 150 biotin molecules can be attached to one antibody molecule,and the strong affinity of the biotin for the glycoprotein avidin allows its use as complex-ing secondary reagents. Biotin labeling of the primary (direct) or secondary (indirect) anti-body can be used in the avidin-biotin methods. In the labeled avidin method the tracer isattached directly to the avidin molecule. In the avidin-biotin bridge method a biotinylatedenzyme such as peroxidase is allowed to bind after attachment of avidin to the biotin-labeled antibody.

In the avidin-biotin (ABC) method a complex of avidin and biotinylated tracercontaining the free avidin binding sites is applied to the biotinylated antibody. As a high

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number of biotin molecules can be attached to a single antibody, a high tracer-to-antibodyratio can be achieved. This property yields high sensitivity and allows the use of anincreased dilution of the primary antibody. The most recently developed and refinedsignal amplification method is termed catalyzed reporter deposition (CARD), which isdiscussed below. The historical development of signal amplification methods has beenpresented by Elias (1999).

Tyramine Amplification Method

The tyramine amplification method is based on the characteristic ability of tyramineto become chemically adhesive following oxidation/radicalization (Gross and Sizer, 1959).Oxidation of the tyramine generates a brown pigment that contains dityramine and moreextensively oxidized and polymerized derivatives. Bobrow et al. (1989, 1991, 1992) usedtyramine for enhancing ELISA, and Adams (1992) adapted it for immunohistochemistry.Subsequently, Mayer and Bendayan (1997) extended the application of the tyramine sig-nal amplification to immunoelectron microscopy (Fig. 4.4). Van Gijlswijk et al. (1997)used green, red, and blue fluorescent tyramides in immunohistochemistry, immunocyto-chemistry, and fluorescence in situ hybridization. Other applications of tyramine includeWestern blotting (Wigle et al., 1993) and in situ hybridization (Kerstens et al., 1995).Tyramine amplification is an important development in advancing the efficiency ofimmunohistochemistry, and its further applications are expected.

The catalyzed reporter deposition (CARD) amplification signal method was initiallydescribed by Bobrow et al. (1989). It is based on the deposition of biotinylated tyramineat the location of the probe catalyzed by horseradish peroxidase (HRP). It has been estab-lished that the highly reactive intermediates formed during the HRP-tyramide reaction willbind to tyrosine-rich moieties of proteins present in the vicinity of the HRP binding sites.The binding of tyramine to proteins at the site of HRP occurs via the production of freeradicals by the oxygen liberated by HRP. In other words, HRP reacts with and thephenolic moieties of tyramine to produce a quinonelike structure bearing a radical on theC2 group. Because this reaction is very short lived, deposition occurs only in the locationat or in immediate proximity to where it is generated. The biotin conjugated to the boundtyramine is subsequently used for the attachment of avidin, which is conjugated to HRP.This HRP is then used to catalyze the brown color reaction. This method allows high-resolution detection of primary antisera because the tyramide complex precipitates only atthe site of reaction. Toda et al. (1999) have compared the immunostaining using conven-tional avidin-biotin complex (ABC) with tyramide signal amplification-avidin-biotin com-plex (TSA-ABC); relatively distinct staining was apparent in the latter technique.

As the TSA-ABC protocol dramatically improves the signal intensity by the peroxidase-catalyzed deposition of biotinylated tyramide, blocking of endogenous peroxidase is required.To ensure quenching of the residual peroxidase, the use of a higher concentration and dura-tion of treatment with is recommended.

The amplification power of TSA-ABC can be enhanced by using several subsequentcycles of incubation or by extending the duration of incubation, for example, up to 30 minat 37°C. Longer incubations may result in background noise (nonspecific staining). Also,the number of cycles possible before the background noise level becomes unacceptable is

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limited to two or three. It should be noted that because the tyramide deposition reaction israpid, small differences in amplification duration may lead to variations in the final signalintensities. Furthermore, because this reaction amplifies both specific and nonspecificimmunohistochemical signals, it is essential that appropriate positive and negative controlsbe used to achieve correct interpretation of staining. Also, because TSA-ABC tends toenhance the background noise along with the signal, the procedure must be optimized toensure low nonspecific binding. All tyramide conjugates yield approximately the same

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results, indicating that signal amplification is independent of the tyramide conjugate used(Speel et al., 1998). Numerous biotin conjugated tyramides can be detected with avidin-conjugate (Totos et al., 1997). If biotin reaction fails, the primary reason is the age of thebiotin solution. Because the shelf-life of newly synthesized biotin is not known, one shouldat least be aware of the expiration date of the reagent.

Different authors and commercial suppliers have assigned different names to signalamplification using tyramine. For example, tyramine signal amplification (TSA) systemand the catalyzed signal amplification (CSA) system are commercially available fromDuPont NEN Life Science Products, Boston, MA, and DAKO Corporation, Carpinteria,CA, respectively. In addition, the terms CARD (catalyzed reporter deposition) (Bobrowet al., 1989), TA (tyramide amplification) (Shindler and Roth, 1996), and ImmunoMax(Merz et al., 1995) have been used for the tyramine amplification technique. The use ofdifferent names for almost identical procedures has resulted in confusion. To standardizethe terminology, the neutral abbreviation, tyramide amplification technique (TAT) shouldbe accepted (Von Wasielewski et al., 1997).

One of the variations of the tyramide amplification technique is termed ImmunoMax(Merz et al., 1995). In this approach the biotinylated tyramine enhancement is combinedwith an antigen retrieval method such as microwave heating, enzyme (proteinase K) diges-tion, or exposure to a detergent (guanidine hydrochloride). This method is effective indetecting some previously unreactive, inadequately reactive, or partly demasked antigensin the formaldehyde-fixed and paraffin-embedded tissues. It has been claimed that thistechnique allows as much as 10,000-fold dilution of the primary antibody and 100 to1,000-fold increase in sensitivity compared to those used with the conventional ABCmethod (Merz et al., 1995). However, the sensitivity increase in the range of 5- to 50-foldis more feasible (Speel et al., 1999).

Preparation of Biotinylated Tyramine

One hundred milligrams of sulfosuccinimidyl-6- (biotinimide) hexanoate (NHS-LC-biotin) (Pierce, Rockford, IL) is dissolved in 40 ml of 50 mM borate buffer (pH 8.0). Tothis solution is added 30 mg of tyramine hydrochloride (Sigma Chemical Company, St.Louis, MO). The solution is stirred overnight at room temperature and filtered (filter). The final biotinylated tyramine concentration is Before application, thesolution is diluted 1:160 in Tris-HCl buffer (pH 7.6) containing 0.03%

Rolling Circle Amplification

Although immunohistochemistry is a versatile and powerful tool for various molecu-lar and cellular analyses, especially for antigen detection, it has a few limitations, such aslack of standardization and difficulty in visualization of antigens present at low concen-trations. In numerous instances important biological markers for cancer, infectious disease,and biochemical processes are present at too low a concentration in tissues or body fluidsto be detected by conventional methods. The difficulty of detection of low concentrationsof antigens can be minimized by antigen retrieval using heating or enzymatic digestion.

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This problem can also be lessened by using stronger fluorochromes and chemiluminescentsubstrates for use in ELISAs, immunofluorescence-based staining, and immunoblotting.

Detection of low concentrations of antigens can also be achieved by increasing thesignal without raising the level of nonspecific background staining. Signal amplification,for example, can be achieved by successive steps of enzymatic reactions. Biotinyl tyramideis commonly used to increase the signal of low abundance targets that are otherwise unde-tectable by conventional techniques. However, tyramide-based amplification may increasebackground noise because of multiple steps of signal amplification (discussed in this chap-ter). Therefore, molecular tissue pathology requires techniques of greater sensitivity andspecificity. One of such techniques to refine the examination of cell components is rollingcircle amplification (RCA) discussed below (Lizardi et al., 1998).

Rolling circle amplification is essentially a surface-anchored DNA replication thatcan be used to visualize single molecular recognition events. It is an isothermal nucleicacid amplification protocol that differs in several aspects from the polymerase chain reac-tion (PCR) and other nucleic acid amplification methods. The RCA can replicate circular-ized oligonucleotide probes with either linear or geometric kinetics under isothermalconditions. It has sufficient sensitivity to detect individual oligonucleotide hybridizationevents and single antigen-antibody complexes (Schweitzer et al., 2000). The linear modeof RCA can generate signal amplification during a brief enzymatic reaction.Another advantage of linear RCA is that the product of amplification remains connectedto the target molecule. Signal amplification by RCA can be coupled to nucleic acidhybridization and multicolor fluorescence imaging to detect single nucleotide changes inDNA within a cytological context or in single DNA molecules (Zhong et al., 2001). Thisprotocol has been used for visualizing target DNA sequences as small as 50 nts long inperipheral blood lymphocytes or in stretched DNA fibers (Zhong et al., 2001).

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Chapter 5

Problems in Antigen Retrieval

LACK OF IMMUNOSTAINING

The failure of antibodies to immunostain tissues or cells does not necessarily reflect theabsence of epitopes. Lack of, or reduced, immunostaining can be attributed to multiple fac-tors. The most common factor is the inability of the antibody to reach and recognize theepitope under the preparatory conditions used, including fixation, dehydration, embed-ding, deparaffinization, rehydration, and incubation. The inaccessibility of the epitope tothe antibody may be due to the formation of large, compact protein complexes as a resultof crosslinking by formaldehyde. These complexes create a barrier to antibody penetration.This aspect of immunostaining failure is elaborated upon later. It is also possible that theantigen molecule is folded and thus hides, the epitope, especially from monoclonal anti-bodies. Apparently, better immunostaining depends on improving antibody access to, andrecognition of, the epitope.

It is well established that many types of antigen molecules are altered by dehydrationsolvents and other reagents. Lack of immunostaining may also be due to excessivelydiluted antibody, to loss of antibody owing to degradation by bacteria or fungi, or to anti-body aggregation due to repeated freezing and thawing. Finally, a monoclonal antibodywill not recognize an epitope in vivo if the former is raised against a denatured antigen.This is also true for polyclonal antibodies when the recombinantly produced antigenbecomes denatured during isolation and purification (Binder et al., 1996).

Fixation with aldehydes plays a key role in the two above-mentioned events: antibodyaccess to and recognition of the epitope. Tissues and cell cultures are usually fixed with analdehyde prior to immunostaining. Fixation has the advantage of anchoring in situ anti-gens, as aldehydes are powerful protein crosslinking agents. They crosslink proteins andglycoproteins through reversible and irreversible alterations in the molecular conformationof proteins, including antigens (epitopes). If the change in conformation is stronglyirreversible, the antibody will have difficulty recognizing the altered epitope, especiallyaldehyde-sensitive antigens. This problem is especially acute when specimens are fixedwith glutaraldehyde. This effect of aldehydes on epitopes and their surrounding proteins iscalled epitope masking. Briefly, epitope unmasking can be accomplished by weakening orbreaking down the protein crosslinking introduced during aldehyde fixation and allowingthe epitope to be exposed to the antibody, provided the latter has access to the former.Thus, two simultaneous events (unmasking of epitope and access of antibody to epitope)

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must take place to achieve immunostaining. Detailed methods of unmasking or retrieval ofepitopes after fixation with an aldehyde are discussed later.

Note that there is no single, ideal pretreatment for retrieving all types of epitopes; theoptimal treatment for a given epitope may not be effective for another type of epitope. Thisis particularly true when considering the pH of the epitope retrieval solution and the dura-tion of pretreatment. These and other pretreatment conditions are explained later. Note alsothat less than optimal processing conditions for a given epitope may also result in back-ground immunostaining.

BACKGROUND STAINING

Background staining is one of the common problems in immunohistochemistry, andit has a number of causes, which are discussed below. One cause is protein hydrophobic-ity, which can occur between different protein molecules. Fixation with aldehydes rendersproteins more hydrophobic as a result of crosslinking between reactive amino acids. Thecrosslinkages are both intramolecular and intermolecular (Hayat, 2000a). The extentof hydrophobic crosslinking depends on the duration, temperature, and pH of fixation.Because changes in these factors result in variable hydrophobicity, owing to variableprotein crosslinking, once an optimal fixation is determined, it must be maintained andcontrolled. According to Boenisch (2001), excessive background staining resulting fromoverfixation with formalin can be remedied by postfixation with Bouin’s or B5 fixative.It should be noted that the greater the proximity of the pH of the antibody diluent and theisoelectric point (pI) of the antibody, the stronger the hydrophobic interaction. In contrast,the lower the ionic strength of the diluent, the weaker the strength of hydrophobic attraction.Hydrophobic interactions can also be reduced by adding a detergent, such as Tween 20, tothe antibody diluent.

The best approach to significantly reduce background staining due to hydrophobicinteraction is to use a blocking protein immediately before or also during the applicationof the primary antibody. The blocking protein must be of the type that competes effectivelywith IgG for hydrophobic binding sites in the tissue. Also, the blocking protein should beidentical to that present in the secondary link or labeled antibody, but not that in the pri-mary antibody, in order to avoid nonspecific binding of the secondary antibody. To fulfillthese requirements, 1% bovine serum albumin (BSA) is added to the primary antibodydiluent. Nonfat dry milk or casein can be used in place of BSA.

The cross-reactivity of antibodies can also cause background staining. This problemarises when the epitope under study is shared among different proteins in the target tissue.Use of polyclonal antibodies can result in nonspecific cross-reactivity with similar or dis-similar epitopes on different antigens. Because an unabsorbed antiserum tends to increasethis problem, it should be subjected to careful affinity absorption. Use of antibodiesfrom hyperimmunized animals will also help. Careful screening of clones in the case ofmonoclonal antibodies will eliminate this type of background staining. Antibody cross-reactivity has been discussed in more detail in Chapter 2.

The presence of even small amounts of natural antibodies in the serum may also pro-duce nonspecific staining. These antibodies result from prior environmental antigenicstimulation. In fact, such antibodies may increase in the titer during immunization of the

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animal with adjuvants. Although these antibodies are difficult to remove, their net effectcan be almost eliminated by using the antiserum at a sufficiently high dilution or by reduc-ing the duration of incubation.

Nonspecific staining may also result from contaminating antibodies produced by thehost’s immune system as it reacts to isolated antigens used for immunization (Boenisch,2001). Isolated antigens are rarely pure. If these antibodies are a problem, the antiserumshould be subjected to affinity absorption. Fortunately, such antibodies are present in avery low concentration and may not cause troublesome background staining. Use of high-titered antisera at sufficiently high dilutions would eliminate this problem. Natural andcontaminating antibodies do not cause any problem when using monoclonal antibodies.

Nonspecific staining can be caused by Fc receptor glycoproteins present on the cellmembrane. This problem is more relevant to frozen sections and smears than to tissuesfixed with formaldehyde. The problem can be avoided by using fragments insteadof whole IgG molecules (Boenisch, 2001). Complement-mediated binding may also causebackground staining in frozen sections when whole antisera is used; however, this prob-lem is not very common.

Antigen diffusion can cause specific background staining. This problem arises whenthe target antigen is displaced from its site of synthesis or storage. Delayed fixation and/orincomplete fixation with formaldehyde tend to cause this problem. Optimal fixation withthis monaldehyde anchors the antigens at their site of synthesis. Mechanical injury to thetissue or drying of the tissue prior to fixation may result in diffuse background staining.Necrotic areas due to autolysis of the tissue tend to stain with almost all staining reagents.Antigen retrieval with prolonged enzyme digestion often disrupts cell architecture, result-ing in the displacement of target antigens from their site of greater density; the net effectis increased background staining.

Background staining also results from the presence of endogenous peroxidase in theformalin-fixed tissues. This artifact can be avoided by treating the tissue sections with 3%hydrogen peroxide in water for 4–9 min at room temperature; methanolic hydrogen per-oxide is not recommended. Blocking of the endogenous peroxidase activity is especiallydesirable with cell preparations and frozen sections (Boenisch, 2001).

Endogenous biotin, distributed in a wide variety of tissues, may also cause back-ground staining with biotin-based immunohistochemical techniques. This biotin is espe-cially abundant in liver, whereas it is poor in the central nervous system and adipose tissue.Endogenous biotin activity is more abundant in the cytoplasm and cryostat sections but isalso present in sections of paraffin-embedded tissues. This problem is largely eliminatedby using streptavidin-based methods or by sequential treatment of sections (prior to stain-ing) with 0.01–0.1% avidin followed by 0.001–0.01% biotin for 10–20 min each. Thebiotin problem is discussed in more detail later in this chapter.

Other causes of diffuse background staining include the presence of residual embed-ding medium and bacterial or yeast contamination in the water bath. The presence ofundissolved chromogen granules on occasion may create the problem of nonspecific stain-ing. Excessive counterstaining with reagents, such as hematoxylin and eosin, may com-promise specific staining.

Finally, a few published reports indicate that antigen retrieval at extremely high tem-peratures may result in nonspecific staining. Baas et al. (1996), for example, have reportedfalse-positive results at a very high antigen retrieval temperature using monoclonal

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antibody D07 against p53 antigen. They carried out antigen retrieval at 96°C for 30 min inthe Target Unmasking Fluid (TUF) containing 35% urea in a microwave oven. This com-bined treatment is unusually excessive and is not used routinely.

PROBLEM OF ENDOGENOUS BIOTIN

The presence of endogenous biotin is a known potential source of nonspecific stainingin immunohistochemical methods based on the avidin-biotin system (Fig. 5.1/Plate 3A andB). Biotin is a water-soluble monocarboxylic acid (a vitamin) of molecular weight244 Da in living cells. This vitamin functions as a prosthetic group for carboxylase enzymesused in fatty acid biosynthesis and gluconeogenesis. It is widely distributed in many tissuetypes, including liver, kidney, breast, pancreas, salivary glands, skeletal and cardiac mus-cles, adipose tissue, and a variety of neoplasms (e.g., salivary gland neoplasm [Lu et al.,2000]). Biotin has been demonstrated immunohistochemically, for example, in human thy-roid, parathyroid, adrenal, salivary, mammary, and prostate glands (Green et al., 1992). Thepresence of cytoplasmic endogenous biotin has also been demonstrated in thyroid papillarycarcinoma (Kashima et al., 1997). This vitamin is found both in the cytoplasm and in themitochondria.

The avidin-biotin complex (ABC) method and the streptavidin-biotin (SAB) methodare more sensitive than the peroxidase-antiperoxidase (PAP) method for histochemicaltechniques. The strong noncovalent attraction between biotin and avidin or streptavidinis exploited in many histochemical, immunohistochemical, and in situ hybridization

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procedures discussed in this volume. As an example, avidin-peroxidase and streptavidin-peroxidase conjugates and antibiotin antibodies are routinely used for labeling biotinylatedantibodies that bind to antigens. In addition, antibiotin antibodies are employed in multi-step protocols to enhance the sensitivity of immunohistochemical and in situ hybridizationmethods (McQuaid and Allan, 1992).

However, the avidin-biotin and the streptavidin-biotin detection systems in some casescan lead to false-positive immunostaining. The avidin conjugates employed in these biotin-avidin methods bind to the endogenous biotin, thereby resulting in artifactual staining. Suchspurious staining has been documented in the above-mentioned tissues as well as in such dis-parate tissues as gestational endometrium and ovarian lipid cell tumors (Seidman et al.,1995). Another example of false-positive immunostaining is the purported presence ofinhibin in hepatocellular carcinomas (McCluggage et al., 1997). However, a recent study hasdemonstrated that when endogenous biotin is blocked, immunostaining of inhibin in hepato-cellular carcinomas and hepatocytes is absent (lezzoni et al., 1999). By blocking endogenousbiotin, highly specific staining of endothelium using CD-34 as an antibody without nonspe-cific tubular staining has been achieved in the kidney tissue (Rodriguez-Soto et al., 1997).

The frequency of such erroneous staining is likely to increase as the sensitivity of theprotocols for labeling biotin improves (i.e., biotin amplification techniques) (Adams,1992). It is thought that the problem of endogenous biotin staining is more serious withsome antibiotin antibodies than with streptavidin conjugates (Cooper et al., 1997). Theproblem depends on the affinity/sensitivity of the antibody used. Also, the problembecomes more prevalent when tissues are pretreated with detergents or digestive enzymesfor antigen retrieval (Satoh et al., 1992). Moreover, the intensity of this artifact is enhancedby heat-induced antigen retrieval methods. This nonspecific staining is also observed inin situ hybridization with biotinylated probes (Kashima et al., 1997).

The presence of this artifact poses a distinct risk of its being interpreted as positivestaining, as the artifact can be intense and may be precisely located in the cells of interestwith a clean background. It is known that liver and kidney can retain high amounts ofretrievable biotin-avidin activity in neoplasms. The need for adequate controls or biotin-blocking procedures is obvious when histochemical or immunohistochemical proceduresare used. Negative controls facilitate identification of such nonspecific staining.

An alternative to the avidin-biotin technology, the EnVision™+System (Dako) detec-tion method, is recommended for universal use in diagnostic and research studies. It is basedon enhanced polymer methodology. In comparison with APAAP, PAP, ChemMate™, CSA,LABC, and SABC methods, the En Vision™+System yields optimal detection (Sabattiniet al., 1998). Its sensitivity is at least as good as that of Strept ABC techniques, and its usecompletely eliminates the problem of endogenous biotin.

Another more recently introduced method to prevent endogenous biotin staining con-sists of using a nonbiotin amplification (NBA) detection system (Zymed, San Francisco,CA) (Shi et al., 2000b). This method is as effective as the conventional technique using theLab-SA kit (Histostain-Plus kit, Zymed) but avoids nonspecific biotin staining. The NBAkit is composed of an FITC-labeled secondary antibody and a horseradish peroxidase–conjugated anti-FITC antibody. Figure 5.2 (Plate 1F and G) shows HER-2 staining in theinfiltrating ductal carcinoma cells of breast using the Lab-SA kit or the NBA kit. If eitherof the two methods mentioned above is not used, the following procedure can be employedto avoid endogenous biotin staining.

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Endogenous biotin-avidin activity can be blocked by treating the sections of formalin-fixed and paraffin-embedded tissues with avidin solution alone or followed by biotinsolution. These treatments are carried out after heating for antigen retrieval, and they do notinterfere with subsequent immunoperoxidase staining. The concentration of the avidin iscritical in blocking endogenous biotin. A cost-effective alternative to commercially avail-able avidin, which is rather expensive, is dilute egg white (Miller and Kubier, 1997). Avidinsolution can be prepared by mixing two egg whites in 200 ml of distilled water. Skim milkshould not be used as a substitute for commercially available biotin (R.T. Miller, personalcommunication). Use of 0.2% biotin in PBS is recommended. The specificity of thismethod requires the absence of both avidin-binding sites and peroxidase activity in the tis-sue sections. Immunoperoxidase staining of endogenous biotin in frozen sections can beeliminated by their treatment with 1% hydrogen peroxide in methanol (Cooper et al.,1997). Such treatment should be applied after application of primary antibody.

Procedure (lezzoni et al., 1999)

Sections ( thick) of formalin-fixed and paraffin-embedded tissues are placed onslides, dried overnight at 37°C, deparaffinized with xylene, and rehydrated with descendingconcentrations of ethanol. They are treated with 3% hydrogen peroxide in methanol for 5–10 min and then rinsed with PBS. The slides are immersed in 10 mM citrate buffer (pH 6.0),and heated for 2 min at 100% power, followed by 8 min at 80% power. After being rinsedin PBS, the sections are incubated in an appropriate primary antibody. They are rinsed inPBS and then treated for 4–8 min with egg white avidin solution (2.5 g of egg white avidinin 0.1 mM PBS) (Ventana Medical Systems). Avidin binds to endogenous biotin.

Following rinsing in PBS, the sections are treated with free biotin solution (2.5 mgin 0.1 mM PBS) for 4 min to saturate the remaining binding sites of the egg white avidin.The sections are thoroughly rinsed with tromethamine-based buffer or PBS, followedby the following sequential immunostaining protocol: biotinylated secondary antibody,

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avidin-strep+avidin-horseradish peroxidase conjugate, DAB-hydrogen peroxide, copperenhancer, and hematoxylin. The slides are dipped in lithium carbonate, and cover-slipped.

MIRROR IMAGE COMPLEMENTARY ANTIBODIES

The conventional immunodetection system may contribute to poor staining specificitybecause of unwanted interactions between the immunoreagents and the endogenous tissuecomponents. Avidin-biotin-based systems, for example, may cause unwanted stainingby reacting with endogenous biotin. Another example is the presence of the endo-genous enzyme, which tends to cause nonspecific chromogen precipitation. To avoid some ofthese problems, Mangham and Isaacson (1999) introduced a novel and sensitive peroxidase-based immunohistochemical detection method that employs mutually attractive, mirrorimage complementary antibodies (MICA). Such antibodies consist of two polyclonal anti-bodies raised in different species that are mutually attractive, i.e., they are raised againsteach other’s immunoglobulin species. Thus, each antibody is both an antigen and an anti-body with respect to the other.

Compared with the ABC technique, the MICA method allows up to 200-fold dilutionof the primary antibodies with equivalent or superior immunostaining and shorter dura-tions of incubation. Other advantages of the MICA method are that it is avidin-free andthus avoids nonspecific staining due to endogenous biotin, and yields ~64-fold increase insensitivity (as judged by dot-blot) compared with that of the ABC technique. The improvedsensitivity of the MICA protocol is thought to be due to increased stability of the com-plexes produced and possibly to antigen bridging. A minor limitation is the longer dura-tion required to complete this method.

Procedure

Freshly paraffin-embedded or archival paraffin-embedded tissues can be used(Mangham and Isaacson, 1999). Sections ( thick) are deparaffininzed in xylene andrehydrated in descending concentrations of ethanol. Antigen retrieval is carried out byheating the sections in 1 mM EDTA (pH 8.0) in a pressure cooker at full pressure for 2 min.The sections are allowed to cool and are then transferred to TBS (pH 7.4). They are treatedwith 3% in methanol for 15 min to block endogenous peroxidase activity andwashed in TBS/Tween ( Tween/1 ml TBS). The sections are incubated in the primaryantibody (diluted in TBS) for 1 hr and then washed in TBS/Tween. They are incubated in1:30 diluted link antibody (sheep antimouse Ig or sheep antirabbit Ig) (the MICA targetantibody) for 20 min, and then washed in TBS/Tween.

The sections are incubated in 1:30 diluted MICA antibody No. 1 (peroxidase-conjugated donkey antisheep Ig) for 20 min and washed with TBS/Tween. They are incu-bated in 1:30 diluted MICA antibody No. 2 (sheep antidonkey Ig) for 20 min and washedin TBS/Tween. This is followed by incubation with 1:30 diluted MICA antibody No. 1(peroxidase-conjugated donkey antisheep Ig) for 20 min and washed in TBS/Tween. Thesections are exposed to 1 mg/ml DAB-0.02% in TBS for 6 min and washed in TBS.They are dehydrated in ethanol, counterstained with hematoxylin, and cover-slipped.

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All the reagents are commercially available in kit form (polyMICA: Binding SiteLtd., Birmingham, UK).

FIXATION OF FROZEN TISSUES

Occasionally, tissues frozen for intraoperative consultation are processed forimmunostaining. This situation arises, for example, when lesional tissue or atypical cellsthat are seen in a frozen section require confirmation. Some antigens show decreased ornegative staining in tissues that are frozen before fixation with formalin, whereas the stain-ing of some other antigens remains unchanged. For example, the staining of S-100, HMB-45, synaptophysin, and neuron-specific enolase was negative in frozen tissues that weresubsequently fixed with formalin, but the staining of these antigens was positive in fresh-formalin-fixed tissues (Edgerton et al., 2000). The staining of chromogranin was decreasedin frozen-fixed tissues. In contrast, the staining of cytokeratins remained unchanged infrozen-fixed tissues. This and other evidence indicates that sections of frozen tissue thathave been subsequently fixed with formalin may show false-negative staining.

Although the exact mechanism responsible for the above-mentioned false-negativestaining is not known, it is likely that cell membranes are disrupted when frozen tissue isthawed during fixation. Such a disruption does not occur when fresh tissue is fixed. Thus,damaged membranes would facilitate antigen diffusion out of the nucleus and/or the cell.This problem is especially serious for antigens in neural tissues (Edgerton et al., 2000); there-fore, caution is warranted in interpreting immunohistochemical results of tissues that arefixed preceded by freezing. It is recommended that when surgeons freeze the tissue speci-men, they should also fix freshly cut specimens of the same tissue for comparative study.

Use of frozen sections without postfixation also has limitations in certain cases.The following example testifies to the drawback of using such sections for diagnostic pur-poses. Although intraoperative frozen-section evaluation for the surgical treatment ofHirschsprung’s disease is a common practice, a high rate of incorrect diagnosis of this dis-ease has been reported using frozen sections (Maia, 2000). When surgical pathologists useprimary resection without prior colostomy or frozen sections as the initial diagnostic test,the results of an incorrect frozen section could be disastrous. Because the concordance ratefor frozen-section diagnosis on initial pathological specimens is low (67%), establishingan initial diagnosis of Hirschsprung’s disease on frozen sections is not recommended.Furthermore, the introduction of artifacts tends to make interpretation of already subtlehistological findings untenable. It is therefore recommended that well-prepared permanentsections be used to establish the absence of ganglion cells in a rectal biopsy for the pres-ence of this disease. It is known that pathological diagnosis of Hirschsprung’s disease isestablished by demonstrating the absence of ganglion cells in the colonic neural plexuses.

HOT SPOTS (AREAS) IN MICROWAVE OVEN

Microwaves consist of electric and magnetic fields, and they propagate in space.Electric fields are primarily responsible for physical effects on the tissue. The energy dis-tribution and thus the speed of absorbing energy and warming up vary topographically. In

Problems in Antigen Retrieval 103

other words, the spatial energy distribution in a microwave oven is unequal. A region inthe oven with a high intensity of electromagnetic fields is known as a hot spot or, more cor-rectly, a hot region (~1 cm).

Hot areas are not located at fixed coordinates in the oven and are influenced by theload placed in the oven. Loads of various sizes and shapes lead to different heating pat-terns. By varying the position of the load within the oven during irradiation, reproducibleresults can be obtained. The problem of hot areas can be solved by providing a microwavetransparent rotating platform for the load during irradiation and by placing an extra load toserve as a heat sink; a jar containing 100 ml to 1 liter tap water or antigen retrieval solu-tion suffices. To obtain reproducible results the same type of jar should be placed in thesame location in the oven. For detailed theoretical and practical considerations of hotareas, the reader is referred to Kok et al. (1993).

PROBLEM OF ANTIGEN RETRIEVAL STANDARDIZATION

Lack of reproducibility of results in epitope retrieval immunohistochemistry is aserious problem unacceptable in diagnostic immunopathology. Variable intra- and inter-laboratory results are a common phenomenon. Lambkin et al. (1998) have assessedimmunohistochemical results of estrogen receptor obtained from 16 Irish histopathologylaboratories. They confirmed that although the majority of participants achieved accept-able immunopositive staining in the supplied sections, variations in the intensity ofimmunostaining, focal staining, and nuclear staining were observed. Battifora (1998) hasdiscussed the necessity of minimizing at least intralaboratory variability of results, whichis quite feasible. Both Lambkin et al. (1998) and Battifora (1998) have suggested meansby which some degree of reproducibility of results can be obtained. A large number of fac-tors influence the final results of immunostaining:

1.2.3.4.5.6.7.

8.9.

10.

11.12.13.

Fresh or archival tissue specimensType of fixative and its pH and concentrationDuration of fixation, especially for formalin-sensitive epitopesDuration of tissue storage in formalinEmbedding proceduresType of retrieval fluid and its pHType of microwave oven and heating parameters, other heating sources, or otherepitope retrieval treatments such as enzyme digestion and ultrasoundMonoclonal or polyclonal antibodiesTested or new antibodies obtained from the same clone or notSource of antibodies; even similar monoclonal antibodies obtained from differ-ent sources may show differential affinity for the epitopeIncubation times and temperaturesVariable staining methods; automated immunostainers or manual stainingDifferences in the interpretation of results

Considering the processing variables enumerated above and below, achieving com-plete standardization of immunoreactions is extremely difficult. Standardization of fixationis difficult due to the large number of variables, including the volume and concentration of

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the fixative, temperature and duration of fixation, size and composition of the tissue, and theamount of free blood. Also, irrespective of the size of the tissue block, the fixation is vari-able within the block. The same duration of fixation with formaldehyde may or may notresult in identical fixation, for each tissue block responds uniquely to the fixative.

Moreover, uniform sections are difficult to obtain, and their thickness is difficult todetermine. Many sections are wedge-shaped rather than planoparallel (Rittman, 1998). Inaddition, the arc of vibration caused by the knife edge as it cleaves the section is sufficient,for example, to glide over or under the surface of some nuclei or cut through the remain-der. Consequently, an accurate measurement of the concentration of nuclear antigens isdifficult (Allison, 1999). The tendency is to cut the thinnest possible sections to obtainsuperior resolution that provides distinct images. However, thin sections show excessivecompression as well as variation in thickness because compression is usually inverselyproportional to section thickness. Another obstacle is the small number of sections that areusually examined in a diagnostic laboratory, limiting the production of reliable average orquantitative data.

TEST BATTERY

A standardized method for retrieving a given epitope in a particular tissue can bedeveloped by using the test battery approach (Shi et al., 1996a). This is a convenient andrapid means to optimize three important factors (pH, temperature, and duration of heating)responsible for the immunostaining of a given epitope-antibody combination. The optimalprotocol lessens false-negative immunostaining. The need for this approach arises whenoptimal conditions for retrieving an epitope are not known. It is known that the retrieval ofdifferent epitopes requires specific retrieval conditions. These conditions primarily consistof the pH and the temperature of the epitope retrieval fluid in the microwave oven and theduration of heating. Other factors influencing the final results of immunostaining are notincluded in this test.

The following three levels of heating durations and three pH levels of the epitoperetrieval fluid (sodium citrate buffer and Tris-HCl buffer) have been recommended todetermine the optimal protocol for the retrieval of an epitope (Shi et al., 1996a).

Temperature and duration Buffer pH

120°C for 10 min100°C for 10 min90°C for 10 min

pH1–2Slide # 1Slide # 2Slide # 3

pH 6–8Slide # 4Slide # 5Slide # 6

pH 9–11Slide # 7Slide # 8Slide # 9

One reason for interlaboratory differences in the reproducibility of immunostainingresults is the use of microwave ovens with significant differences in age, power, construc-tion, and design. Individual laboratories should optimize wattages and duration of heatingas well as durations of each of the steps mentioned above; some of them will need to bedetermined by trial and error.

In the absence of standardization, since either false-positive or false-negativeimmunostaining can occur with any antigen retrieval protocol, the effect of the chosenantigen unmasking method on every individual antigen must be determined using careful

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controls. Appropriate positive and negative controls, as well as the study of fresh-frozentissue sections, are required to rule out any false-negative or false-positive staining.Duplicate immunostaining will assess reproducibility.

INTRAOBSERVER AND INTEROBSERVER VARIATION IN DIAGNOSIS

Pathologists play a key role in the diagnosis of cancer, and their histopathologicalassessments are accepted as the gold standard. Although not admitted, the process bywhich a pathologist makes a diagnosis is inherently subjective. A number of factors,including clinical features of the lesion, the clinical impression offered by the surgeon, andthe training and experience of the pathologist, play a part in determining the final “sign-out” diagnosis on which the final treatment decisions depend. These decisions have far-reaching consequences in the quality of health care. It is obvious that no other type of errorin the medical profession is more important, less understood, and less frequently admittedthan fallibility in histopathological diagnosis. Kaugars (1995) has aptly pointed out that thesign-out is written on paper, not on stone tablets.

Can the intraobserver and interobserver variations in diagnosis be eliminated?Unfortunately, the answer is no. However, avoidable fallibilities must be avoided. The inter-observer variations can be significantly reduced by a joint session behind a microscope ina process of “practical agreement” (Vet et al., 1995). Prior to such sessions, participatingpathologists reach consensus on the relevant pathological grading characteristics (theoreti-cal agreement). A theoretical agreement increases the practical agreement between pathol-ogists. Interobserver agreement is definitely improved when pathologists confront eachother’s observations and arguments. Even experienced pathologists will benefit by a jointsession behind the microscope.

Both low-power and high-power microscope observations are useful in markedly min-imizing interobserver variation. The characteristics observed at low magnification includeatypia, location of immature cells, and stratification/polarization. At high magnification,detailed morphological characteristics, such as location of immature cells and stratification/polarization (differentiation), nucleus/cytoplasm ratio, hyperchromasia, polymorphousnuclei (cell characteristics), and the location and appearance of mitotic activity, are scored(Vet et al., 1995).

Another approach to avoiding interobserver and intraobserver variations and standard-izing the diagnosis is the use of computer-assisted analyses. This technology is beginningto be employed in some laboratories (see below).

QUANTITATION OF IMMUNOSTAINING

Accurate quantitation of antigens using immunohistochemistry depends upon a linearrelationship between the amount of antigen and the intensity of immunoperoxidase-DABreaction product as well as the percentage of stained cells. Variations in staining intensitywill reflect the amount of antigen only if optimal preparatory procedures are used; forexample, oversaturation of the chromogen reaction may result in invalid quantitation.Therefore, optimal concentration of DAB should be determined by trials with DAB

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concentrations ranging from 0.1–1.5 mg/ml in Tris buffer containing 0.003%(pH 7.6). Optimal duration of DAB incubation should also be determined by trying incu-bations for 3–15 min at 37°C. Currently, interpretation of immunohistochemistiy stainingin most studies is subjective, qualitative, and nonreproducible.

The achievement of quantitative, reproducible results requires standardized immuno-histochemical procedures. The development of such a universal procedure is difficultbecause antigens are not equally affected by a specific processing protocol, including fix-ation. However, instruments to computerize image analysis have the potential to quantitateimmunohistochemically localized antigens. One such instrument is Cell Image AnalysisSystem SAMBA 4000 (Imaging Products International, Chantilly, VA) used in combina-tion with the automatic stainer (OptiMax Staining System, BioGenex Lab., San Ramon,CA) or Tech Mate (Biotek, Santa Barbara, CA). The image analyzer contains software forthe densitomeric and RGB (red, green, blue) to HSI (hue, saturation, intensity) colorimet-ric analysis of cells and tissues (Esteban et al., 1994a). It is based on a light microscopeattached to an interactive microcomputer that is capable of high-speed digital image pro-cessing for cell measurements.

The system includes various software packages for different applications (De Cresce,1986). It allows immunostained histological sections to be represented as digitized imagesfrom which the optical densities of the DAB reaction product over a specific cell part orcomponent (e.g., nucleus) can be quantitated. Bacus et al. (1988) have successfully quan-tified the estrogen receptor content in human breast tumors. The data showed excellentsensitivity and specificity.

Quantitative immunostaining analysis has been performed with a computerizedmicroscopic image processor, SAMBA 200 (SAMBA TITN, Grenoble, France)(Seigneurin et al., 1987). Integrated optical density histograms are provided by the imageanalysis processor. Mean values and percentage of immunostained cellular surfaces arecomputerized by the application processor. These computerized systems facilitate multi-parametric, accurate, reliable, reproducible, and automatized evaluation of the hetero-geneity of the antigenic sites in tumors (Charpin et al., 1989). The advantage of thiscapability becomes apparent when one considers that tumors showing positive immunos-taining are pools of positive and negative cells.

To rectify the lack of intra- and interlaboratory reproducibility of immunostaining,Riera et al. (1999) have described the Quicgel method used in conjunction with computer-assisted image analysis for quantitation of immunohistochemical data. The Quicgel con-sists of a cultured cell plate containing a known amount of the antigen, which yieldsconsistent positive staining detected by image analysis. The Quicgel is processed simulta-neously with the specimen.

This method is based on the assumption that changes in the antigen content of theQuicgel and the specimen vary in parallel during specimen processing. Thus, a decrease inthe immunostaining of the specimen during processing is equally demonstrated in theQuicgel. Even without image analysis, Quicgel can serve as a control in immunohisto-chemical staining. Further application of this protocol for quantitation is awaited.

A related approach was used by Ranefall et al. (1998) to quantify images of immuno-histochemically stained cell nuclear Ki-67 antigen and cyclin A protein in bladder carci-noma tissue. They combined automatic, computerized image analysis with appropriatecontrols and reference material. This approach is superior to the automatic method without

Problems in Antigen Retrieval 107

controls. The former method consists of simultaneous processing of embedded culturedcontrolled cells and tissue sections. Agarose-embedded cultured fibroblasts are fixed,embedded in paraffin, and sectioned at They are immunostained together with

paraffin-embedded tissue sections.The image of the control cells serves as a standard control regarding image qualities

such as illumination and color properties. Since control cells possess known characteris-tics regarding antigen expression to be examined in tissue sections, they serve as a meansto control and standardize immunohistochemical data (Ranefall et al., 1998).

Recently, an automatic color video image analysis system was developed to quantifyantigen expression (androgen receptor) (Kim et al.,T999a). This system provides a linearrelationship between the antigen content and mean optical density of the immunoperoxidase-substrate reaction product. Titration of antibody, concentration, and reaction duration ofthe substrate can be optimized with this system. The imaging hardware consists of a Zeissmicroscope, a three-chip charge-coupled-device camera, a camera control board, and aPentium-based personal computer.

It is necessary to properly maintain and calibrate the computerized image analysissystem. This is the only way to ensure that the scale of the reported histogram will coverthe range of staining intensities obtained in practice (Bacus et al., 1988). In addition, thetechnician should be knowledgeable about the normal cellular morphology and pathologyof the tissue. Counterstaining tissue sections with methyl green in conjunction with the

chromogen is also necessary.A relevant question is whether the quantitative measurements obtained with currently

available computerized image analysis systems are reliable, accurate, and reproducible andif quantitation of immunostaining reactions offers any real advantage over qualitative eval-uations by an experienced pathologist (Rittman, 1998). Many of the image analysis sys-tems are still somewhat rudimentary. Results obtained with various image analyzers aredifficult to compare because different hardware and software are used. Unfortunately, insome laboratories quantitation of immunohistochemistry may be used simply to justify thepathologist’s decision in difficult (borderline) cases. The controversy over whether bor-derline tumors should be classified as benign or malignant and whether they represent aprecursor of malignancy remains unresolved.

A semiquantitative approach has also been applied for evaluating the concordancebetween the presence of p53 mutations and immunohistochemical overexpression of thisprotein in breast carcinomas (Schmitt et al., 1998). The advantage of this approach is thatit uses a scoring system based on both the intensity and percentage of stained cells. A lim-itation of this method is that scoring is performed by examining all low-power opticalfields containing tumor, which is time consuming, lacks automation, and is thus subjec-tive, even when scored by more than one observor.

AUTOSTAINERS

Presently, immunohistochemistry requires improvements in quality, reproducibility,speed, quantitation, and standardization. Some of these goals can be achieved by usingcomputerized bar code–driven automatic immunostainers that automatically dispensereagents, control washing, mixing, and heating to optimize immunohistochemical reaction

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kinetics and that produce results within 1 hr. Furthermore, the practice of manually staininga large number of slides is tedious and time consuming. Typically, manual hematoxylin-eosinstaining (hematoxylin stains nuclei blue and eosin stains cytoplasm pink) is completed inapproximately 25 steps. Autostainers save a technologist’s time, which permits him or her tocarry out more technically demanding tasks. After the staining protocol has been standard-ized, the stainer does not require the technologist’s attention during its operation.

There are many other advantages to using autostainers. They prevent risk of exposureto certain hazardous reagents (xylene). Some stainers have built-in fume hoods or can beused in a fume hood; in the former case, the need for a large overhead fume hood iseliminated. Another advantage is consistency of the technique that eliminates intra- orinterpersonal variations in results. Also, a consistent temperature can be maintained fortemperature-sensitive procedures. The space requirement for some stainers (e.g., centrifu-gal stainer) is smaller than that required for manual staining for some procedures. Efficientstainers use reagents conservatively, thus reducing the amounts needed and the risk ofcontamination.

There are a few limitations to equipping a laboratory with an autostainer. The highcost of most stainers may be prohibitive for a small laboratory. Unavailability of space insuch a laboratory is also a possibility. Repairs of this machine are expensive and may takea long time if the service technician lives in another state. Some stainers require the pur-chase of prepackaged reagents that are also relatively expensive. Moreover, some technol-ogists may object to being restricted to using these reagents. In addition, a staining defectoccurring during operation of the stainer is revealed only after staining is complete. On theother hand, if a problem arises during manual staining, the process can be stopped and theproblem corrected. But above all, the use of a stainer limits the technologist’s understand-ing of the actual staining process.

There are two types of autostainers: in the first type, the slides are immersed intothe reagent; in the second type, the reagent is applied to the slides. Stainers that immersethe slide into the stain (bath stainers) can be either linear or batch design (Earle, 2000). Thelinear type is based on a carrier mechanism that allows loading of the slides into the slideholders (racks), one at a time, and their sequential immersion into the staining solution.The slide holders are attached to the carrier, which moves at a uniform speed, and theslides exit the stainer one at a time. This type of machine is long, processes~360–720 slides per hour (12–14 min per slide), and requires water and a drain. It mayhave a built-in fume hood and slide dryer. Batch stainers move slide holders, each con-taining several slides, through baths of the staining solution. Programmable batch stainersare now commercially available which use robotic arms to move the slide racks from oneposition to the next. These stainers can be programmed to agitate the slides in the stainingbath. Simultaneous multiple staining can be accomplished in some machines based on thisprinciple. A combined linear-batch stainer is also available, which moves slide containersthrough a series of stain containers. Each rack may hold a small or large number of slides,and continuous staining is possible. The machine is long, processes 24–66 slidesper rack, and the time taken depends on the program. It may be compatible with the cover-slipper, and some have built-in fume hoods. The machine may require running water anda drain, and it may have waste collection.

There are three types of stainers that apply the reagents to the slides: capillary gapstainers, centrifugal stainers, and flat-method stainers (Earle, 2000).

Problems in Antigen Retrieval 109

Capillary Gap Stainers

Capillary gap stainers are based on the principle that the staining solution is forcedbetween the slide and the area around it. Essentially, rotating gears move slides (facedownward) along a plane surface that has holes through which the stain is pumped atappropriate intervals. The advancing slides press a switch as they pass each staining sta-tion, thus activating a pump. The stain is discarded after the slide moves to the next sta-tion, avoiding the contamination of the bulk containers. The machine pumps the stain fromthe closed bulk containers to the plane surface via small tubing, minimizing reagentevaporation.

The capillary gap system can also be used in stainers that use two slides face-to-faceto provide the capillary gap. Robotic arms move holders of paired slides to staining,draining, and rinsing stations. Because this system uses very little staining solution, it isrecommended for immunostaining large numbers of slides. The machine is ~3 ft long,may be compatible with a cover-slipper, and some may have a built-in fume hood. Thissystem requires prepackaged reagents and may require a drain.

Centrifugal Stainers

Centrifugal stainers spray the staining solution onto the slides as they rotate past thespray nozzles in a spinning chamber. The prepackaged reagents are in closed containerswith pump tubing, which prevents evaporation and contamination of the chemicals. Themachine is smaller than 2×2 feet, stains 12 slides in 6–8 min, requires prepackagedreagents, and may require a drain. It usually does not require a fume hood.

Flat-Method Stainers

Flat-method stainers drop staining solutions onto the slide as it lies flat within thestainer. Some stainers employ robotic arms to apply solutions to the slides. This system isin common use for immunohistochemical staining. The machine is long, stains20–40 slides, depending on the system, in and slides require predeparaffinization.The protocol may require prepackaged or manufacturer’s reagents and a waste container.It is recommended for immunohistochemistry. For additional details about autostainers,see Earle (2000).

The following automatic tissue processors and stainers are commercially available.

1.

2.

3.

4.

AP 280 Embedding Station, Carl Zeiss, Inc.One Zeiss Drive, Thornwood, NY 10594ATP1 Tissue Processor, Triangle Biomedical Sciences, Inc.3014 Croasdaile, Durham, NC 27705–47770Cytologix Stainer, Cytalogix Staining System99 Erie Street, Cambridge, MA 02139Lab Vision Auto Stainer, DAKO Corporation,6392 Via Real, Carpinteria, CA 93013

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5.

6.

7.

8.

9.

10.

11.

Mark 5 HSS Stainer, Diagnostic Products Corporation5700 West 96 Street, Los Angeles, CA 90045Medite TST40 Slide Stainer, Mopec13640 Elmira Street, Detroit, MI 48227Medite TPC15 Tissue Processor, Mopec13640 Elmira Street, Detroit, MI 48227Optimax Consolidated Staining System, Biogenex Laboratories4600 Norris Canyon Road, San Ramon, CA 94583Protocal Capillary Action Stainer, Biochemical Sciences, Inc.200 Commodore Drive, Swedesboro, NJ 08085Tissue-Tek. DRS 2000, Slide Stainer, Sakura Finetek USA, Inc.1750 West 214 Street, Torrance, CA 90501Ventana ES Automated Immunostainer, Ventana Medical Systems, Inc.1910 Innovation Park Drive, Tucson, AZ 85737

VOLUME-CORRECTED MITOTIC INDEX

The volume-corrected mitotic (M/V) index can be used to test for differences betweenborderline and malignant tumors. This index expresses mitotic activity as the number ofmitotic figures per square millimeter of neoplastic tissue in the microscope field. Usually10 fields are counted at a magnification of 40, which corresponds to of neoplas-tic tissue in the section. The M/V index has the advantage of not being influenced by thesize variation of the microscope field or cellularity of the neoplasm (Haapasalo et al.,1989). Also, this method is easy, relatively rapid, reproducible, inexpensive, and availableto all pathologists (Miliaras, 1999). The morphometric formula of the M/V index rendersmitotic counts a more reproducible criterion because it avoids some of the limitations, suchas differences in microscope field size, of the conventional mitotic index. The M/V indexhas been used for mitotic counts in many human neoplasms for both diagnostic and prog-nostic purposes (Lipponen et al., 1990). Recently, Miliaras (1999) has used this index fordetermining differences in p53 immunoreactivity and the proliferation rate between bor-derline and malignant ovarian tumors.

The M/V index is evaluated as the number of mitoses per 10 hpf (high power field)and is calculated according to the following formula proposed by Haapasalo et al. (1989).

Where n=number of microscopic fields studied (usually 10)Vv=volume fraction of neoplastic tissue (%) as expressed in the area fraction of neo-

plastic tissue in the microscope field; this is estimated subjectively in the same field inwhich the mitotic count is made.

MI=number of mitotic figures in a microscopic field from the area of highest neo-plastic cellularity (during the measurement, the microscope is focused only once).

k=coefficient characterizing the microscope: where r is the radius of themicroscope field in millimeters (0.255 mm).

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THE GLEASON GRADING SYSTEM

Gleason grading is now the most widely used system for grading prostatic carcinoma.This system is an effective tool for prognostication and as an aid in therapeutic decisionsfor men with prostate cancer. The system is characterized by two major features: (1) it isbased solely on architectural pattern but cytological features are not evaluated (Gleasonand Mellinger, 1974) and (2) the overall grade is not based on the highest grade within thetumor. The prognosis of prostate cancer is intermediate between the most predominant andthe second most predominant pattern of cancer (Fig. 5.3). Consequently, the grades of themost prevalent and the second most prevalent pattern (~5% of the tumor) are addedtogether to obtain a Gleason score (Allsbrook et al., 1999). If the tumor shows only onepattern, the pattern grade is doubled to obtain the Gleason score; for example, for allpattern 3, the Gleason score is 6 (Fig. 5.4). The Gleason score is directly correlated withmortality rates, is a predictor of time to recurrence after surgery, and of response totherapy. Presently, the Gleason score, along with PSA and tumor stage, forms the databaseupon which radical therapies are recommended (King, 2000).

The Gleason score alone suffers from interpretation bias and its accompanying gradeerrors. Evidence is available indicating a lack of interobserver reproducibility of this score.As expected, interobserver agreement is significantly better among pathologists wholearned Gleason grading at a professional meeting or course than among those who had

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not (Allsbrook et al., 2001a, b). The interpretation bias is significantly minimized througha consensus pathological evaluation, while sampling effects are maximally reduced byusing an optimal number of biopsy cores. These two remedies, when applied in combina-tion with the Gleason score, result in maximal grading accuracy. Another approach to

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achieve grading accuracy is through using cytokeratin staining in combination with aconsensus of three pathologists (Carlson et al., 1998).

Although the use of sextant biopsy is an effective technique for prostate cancer diag-nosis (Stamey, 1995), and has now become the most common method, it has limitations.Studies have shown that grading based on sextant biopsies, when compared with matchedsurgical grades, suffers from a significant rate of undergrading. Therefore, biopsy coresshould be outside of the anatomical domain of extant biopsies to reduce missed or delayeddiagnosis (King and Long, 2000).

Because prostate cancer is often multifocal (heterogeneous population of tumorcells), a certain degree of sampling error is expected. The error may result from samplingan area which is either overrepresented with high-grade tumor or, conversely, overrepre-sented with low-grade tumor as compared to the actual histological grade of the resectedprostate (King and Long, 2000). It is not uncommon for well-differentiated cancers to beundergraded and poorly differentiated cancers to be overgraded. To overcome such sam-pling errors, a directed biopsy must be performed (assuming that an ultrasound-visiblelesion is present) or the number and location of biopsies must be increased.

In conclusion, the grading error can be significantly reduced by minimizing samplingeffects through increasing the number and location of biopsies. The positive role of con-sensus pathological evaluation in lessening grading errors is equally important. These tworemedies will improve the accuracy of Gleason grading of prostate biopsies.

As stated above, most patients with prostate carcinoma are diagnosed by core needlebiopsy, and tumors are most commonly graded using the Gleason grading system. TheGleason score assigned by the pathologist to the tumor obtained by needle biopsy can pro-foundly affect the treatment decisions made by urologists, radiation oncologists, and med-ical oncologists. Although presently the Gleason grading is in common use, many studiesindicate inaccuracies in this grading system, with a strong tendency toward undergrading(Carlson et al., 1998).

To improve the accuracy of the Gleason grading system, Kronz et al. (2000) devel-oped a free, web-based tutorial program (www.pathology.jhu.edu/prostate). It consists of20 pretutorial quiz images of prostate carcinoma specimens that were obtained by needlebiopsy for grading, followed by 20 tutorial images with text describing the Gleason grad-ing system. Subsequently, pathologists take a posttutorial quiz, consisting of the same 20images that were used in the pretutorial quiz. This web site tutorial leads to an improve-ment, especially in the grading of high-grade tumors (Gleason score, 8-10). The web sitealso improves the grading of tumors with Gleason scores of 5–6 on needle biopsy. Anadvantage of the web-based media is that it is permanently available for repeated reviewas opposed to other learning experiences such as lectures.

UNIVERSAL ANTIGEN RETRIEVAL METHOD?

Is it practical to develop a universal epitope retrieval method? The answer is no, asthe optimal retrieval of each type of epitope requires very specific processing conditionssuch as fixation, retrieval fluid, and unmasking treatments, including heating, enzymaticdigestion, and ultrasound. In terms of preservation and masking, each type of epitope isaffected differently by the fixative used, and by its concentration, pH, and the temperatureand duration of fixation. As long as different fixatives are used in different laboratories,

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interlaboratory standardization of immunohistochemistry will remain elusive. Storage oftissue in the fixative, as well as sections of the fixed tissue mounted on glass slides, influ-ences epitope retrieval. Some evidence indicates the potential loss of immunostaining instored paraffin sections, especially with prolonged durations at room temperature (seepage 84). It is not uncommon for pathology laboratories of small hospitals to mailunstained slides to other laboratories for immunostaining and second opinions.

Epitope retrieval depends upon epitope preservation and hence on the effects of fixa-tion or chemical fixation. In some cases, the fixation history of archival tissues is notknown, making the rational choice of epitope retrieval methods problematic. Anotherproblem is that similar antibodies obtained from different sources may vary in their sensi-tivity and specificity.

The procedure used to dry sections on a glass slide may also affect the degree ofimmunoreactivity. This is exemplified by nuclear antigens (e.g., proliferating cell nuclearantigen) that show decreased immunoreactivity when sections are hot-plated onto glassslides (Hall et al., 1990). The composition, pH, and amount of retrieval solution influencethe degree of epitope retrieval. In addition, the type of heating used (microwave, autoclave,steamer, hotplate, and conventional oven) affects the degree of epitope retrieval.Overwhelming evidence indicates that all types of epitopes are not equally unmasked withany one source of heating. In other words, certain types of epitopes are maximallyretrieved with microwave heating, while some other types are best retrieved with an auto-clave or a steamer. In addition, ultrasound treatment may be efficient for unmaskingcertain epitopes. Furthermore, optimal retrieval of certain epitopes is obtained withcombined treatments such as microwave heating-ultrasound or enzymatic digestion-microwave heating. Caution is warranted in the use of enzymatic digestions, as this treat-ment can adversely affect cell morphology and antigenicity.

The temperature used during epitope retrieval is important, and if microwave heating isused, so is the amount of water load and its exact location in the oven. The extent of epitoperetrieval is also section-thickness dependent. It should be noted that the same processing con-ditions show different retrieval efficiencies of similar epitopes in animals of different speciesand ages. Whether a protein molecule is glycosylated or not affects its unmasking with anepitope retrieval protocol. For example, it has been demonstrated that the more glycosylatedhuman placenta fibronectin has a higher resistance to protease treatment, and thus a reducedepitope retrieval, than the less glycosylated plasma fibronectin (Zhu et al., 1984). Even dif-ferent isomers of an antigen may require different epitope retrieval methods. In addition, thedegree of epitope retrieval is ethnicity-dependent. Also, optical microscopy, fluorescencemicroscopy, and electron microscopy require different methods of fixation and epitoperetrieval. A pretreatment that facilitates the recognition of a given epitope may destroy otherepitopes in the same antigen or in other antigens. In conclusion, although a universal epitoperetrieval method is almost impossible to formulate, the development of a general strategy foradequate retrieval of epitopes is feasible. Such an approach is presented in Chapter 8.

CALIBRATION OF MICROWAVE OVEN

As stated earlier, lack of a standardized antigen retrieval method results in intra- andinterlaboratory variability in immunostaining results. The following method of microwave

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oven calibration is a step toward obtaining reliable immunostaining (Tacha and Chen,1994). This approach avoids repeated interruption of oven heating to replenish the antigenretrieval fluid. The method essentially establishes the time to boiling point, after which thesetting is adjusted to maintain a simmering temperature. At such mild temperatures, theseparation of sections from the slide is less likely.

Place the jar containing 250 ml of antigen retrieval fluid and slides in the center of themicrowave oven, and set the oven on high power (800 W) for 2–3 min, until the fluidbegins to boil. Turn off the oven and record the exact time it took to achieve boil. Set theoven on low power (~300 W) for 7–10 min, and adjust the setting so that the oven cycleson and off every 20–30 sec and the fluid boils for ~5–10 sec/cycle. Also, note this setting.The following formula can be used to determine the power setting:

S = 250/P × 10,

where S is the oven power setting and P is the output power of the oven. For example,if the oven output power is 800 W, the power setting for antigen retrieval (S) will beS= 250/800 × 10= 3.1. Therefore, set the oven on 3 and heat at 100°C for 7–10 min toachieve antigen retrieval, depending on the antibody used.

Microwave ovens with temperature readouts are commercially available (EnergyBeam Science, Agawam, MA). Their power output is regulated by a temperature feedbackmechanism and timer, so that both temperature and time can be monitored. They can alsobe used for fixation and accelerated immunostaining.

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Chapter 6

Antigen Retrieval

POSSIBLE MECHANISMS OF ANTIGEN RETRIEVAL

Heating, especially microwave heating, effectively unmasks a wide variety of epitopes(e.g., Fig. 6.1). However, in some cases epitopes are best unmasked by methods other thanheating. Such alternate procedures include enzyme digestion and treatment with detergents.Furthermore, the retrieval of some epitopes is accomplished with a combination of methodssuch as microwave heating–enzyme digestion, microwave heating–sonication, or microwaveheating–EDTA. In fact, variations of the heating method may affect epitope retrieval differ-entially. These variations include microwave heating, autoclaving, pressure cooker, waterbath, and even a hot plate. Comparative studies using different heating methods demonstratethat an antigen under study is retrieved optimally by only one of the heating variations, andthat heating method may be other than microwave heating (see pages 153–154). However, itis most likely that each heating method is equally effective in antigen retrieval provided it isused under its optimal conditions. Nevertheless, microwave heating is presently the mostcommonly used procedure to retrieve antigens in formalin-fixed tissues. These observationssuggest that multiple mechanisms are responsible for epitope retrieval. A number of possiblemechanisms responsible for epitope unmasking have been advocated, including breakage ofprotein crosslinks introduced by formaldehyde, denaturation of proteins to reveal previouslymasked epitopes, and unmasking of epitopes by removing calcium ions. Major mechanismsare discussed below.

Fixation with formaldehyde tends to alter the conformation of the protein molecule,making it unrecognizable by the antibody. Antigen retrieval treatments may restore theoriginal, native protein structure, reestablishing the three-dimensional structure of the pro-tein, or coming very close to that state (Shi et al., 2001). In other words, antigen retrievaltreatments may renature the protein structure that was altered during fixation. However,direct evidence supporting the revival of the native conformation of the epitope with anti-gen retrieval pretreatment is lacking.

On the other hand, available evidence supports the occurrence of the breakage of pro-tein crosslinks, which allows the antibody access to the antigen. Conventional heat, dry orsteam, breaks down reversible protein-protein, protein–nucleic acid, and protein-carbohy-drate crosslinks introduced by formaldehyde and thereby unmasks the epitopes, as well asallowing the antibodies access to the epitopes. It is well known that most, if not all,crosslinks formed during formaldehyde fixation are destroyed upon heating even at 37°C

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for a prolonged time (e.g., 48 hr) or at higher temperatures (e.g., 80–100°C) for very shortdurations, that is, in minutes. Specific peptide-bond cleavage by microwave heating inweak acid solutions is a well-established method in protein chemistry (Wu et al., 1992).The specific cleavage sites of peptide bonds are located at the carboxyl- and amino-terminalends of aspartyl residues along the peptide chain. Thus, heat can free epitopes from otherproteins and attached molecules.

That heat is not the only method to unmask epitopes is exemplified by enzyme diges-tion or detergent treatment. The exact mechanism responsible for epitope retrieval with ultra-sound is not clear, although intense heat is produced for an exceedingly short duration. It isknown, however, that ultrasound and/or heat decreases the amount of negative charges on thecell surface (Joshi et al., 1983; Adler et al., 1988). Mechanical vibrations of molecules causedby ultrasound and heat are thought to unfold the protein molecule and to expose the epitopes.

The mechanism underlying unmasking of epitopes with digestive enzymes is betterunderstood. Enzymes such as trypsin II, used in epitope retrieval, are powerful, testedprotein-digestive molecules. They are known to digest proteins and break down proteincrosslinkages introduced during formaldehyde fixation. As a result, the tight network sur-rounding the epitopes is dismantled, allowing access of antibodies to the epitopes. If anti-gen retrieval with protease digestion must be carried out, 100 mg of trypsin in 100 ml ofTris-buffered saline (pH 7.8) can be used for 15–20 min at 37°C.

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Some information is available on the possible mechanism underlying masking ofepitopes in the presence of calcium ions. Such a masking of epitopes and their unmaskingare discussed in detail on page 120.

Nonthermal Effects of Microwave Heating

Like conventional heat, microwave heat breaks down protein crosslinks. The mostcommon explanation presented in the literature for unmasking epitopes with heat in themicrowave oven is hydrolysis of protein crosslinkages. However, the effects of microwaveheating on the tissue sections resulting in epitope retrievals are exceedingly complex. Heatalone may not be enough to explain the effects of microwaves on epitope retrieval. Thisview is supported by the observation that microorganisms are killed at lower microwavetemperatures or with shorter exposures than those required when conventional heat is used(Chipley, 1980). Furthermore, because microwave heating also enhances immunostainingof ethanol-fixed tissues, it is apparent that such heating unmasks epitopes by a mechanismother than or in addition to breakage of protein crosslinks because this coagulative fixativedoes not introduce crosslinks.

The above-mentioned evidence indicates that in addition to the direct thermal effectof microwaving, microwave heating facilitates epitope retrieval by another simultaneousmechanism. The microwave energy irradiated on the tissue sections in various liquid mediais lost or absorbed by the samples by two mechanisms: ionic conduction and dipole rota-tion. Both effects occur simultaneously to account for the phenomenon of rapid heating(Kingston and Jessie, 1988). It is thought that microwaves unfold protein molecules,exposing the epitopes by subjecting the molecules, at least polar molecules such as waterand polar side chains of proteins, to rotational movement. As a result, these moleculesreach to a high energy level, unmasking the epitopes.

It is known that microwaves interact with dipolar molecules by (1) imparting kineticenergy and raising temperature and (2) altering electric fields. Microwaves induce dielec-tric fields, causing dipolar molecules to rapidly oscillate 180 degrees. In other words, thesemolecules oscillate at the frequency of 2,450 MHz or at about 2.5 billion cycles persecond. Thus, microwave action is also due to rapid oscillation along the axes of asym-metrical molecules such as water, proteins, and fatty acids, which behave as dipoles in anattempt to reorient their positive and negative poles to keep up with the rapidly changingelectrical fields generated by microwaves (Salvatorelli et al., 1996). It has been shown thatthe oscillating electric field causes cell poration (Chang, 1989). It is also known thatmicrowaves irreversibly alter the plasma membrane, with subsequent changes in ion trans-port, breakdown of hydrogen bridges and secondary bridges, alterations in protein hydra-tion, and release of bound water. All of these phenomena explain why microwaves exert adifferent effect than that of conventional heat. It is apparent that not only the thermal butalso the nonthermal component of microwaves deserve consideration as effective epitoperetrieval factors.

Evidence indicates that microwaves affect the kinetics of conformation changes ofproteins such as (Bohr and Bohr, 2000). It is thought that even approxi-mately a few GHz can excite protein molecules. Consequently, the kinetics of conforma-tional changes of the protein molecule are enhanced, and this denaturing effect is

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nonthermal. In fact, microwave irradiation can cause folding or unfolding of protein mol-ecules. However, additional evidence is needed to substantiate the role of the nonthermaleffect of microwave irradiation in antigen retrieval.

EFFECT OF ENDOGENOUS CALCIUM ON ANTIGEN MASKING

An understanding of the interaction between the antigen (epitope) and the antibodyvisualized immunohistochemically can be attempted by considering at least the role offormalin fixation, antigen retrieval fluid, and heat treatment. How each of these three factorsaffects the molecular structure of the antigen or the epitope is the fundamental question. Thesignificance and relevancy of this question are apparent because an antibody recognizesthe corresponding epitope based on the molecular structure of the latter. The following dis-cussion considers the possible role of calcium in masking antigens during fixation withformaldehyde; it is based primarily on studies carried out by Morgan et al. (1994, 1997a,b), Shi et al. (1997, 1999a), and Taylor et al. (1996a, b). The role of other factors in anti-gen masking and retrieval is discussed elsewhere in this volume.

Endogenous calcium is an important factor in epitope masking. Biochemical studiesindicate that calcium binding induces a conformational modification of the protein mole-cule, resulting in either a reduced antigen-antibody recognition effect (e.g., for throm-bospondin) (Wilson, 1991) or the reverse effect (for protein C, a vitamin K–dependentenzyme involved in blood coagulation) (Wakabayashi et al., 1986). It has been proposedthat removal of calcium by chelation significantly modifies the thrombospondin confor-mation (Dixit et al., 1986). These changes may expose epitopes necessary for the bindingof certain monoclonal antibodies. This and other evidence indicates that calcium-inducedchanges in the conformation of different proteins may result in negative or positive detec-tion of immunogenicity. The ability of some monoclonal antibodies, but not all, to recog-nize their corresponding epitopes is calcium-dependent under certain conditions. Differentantibodies respond differently to the calcium-induced modification of the same protein.

In relation to fixation with an aldehyde, possible mechanisms responsible for mask-ing or unmasking epitopes as a result of tissue-bound calcium and calcium chelation,respectively, are detailed below. One of the major effects of formaldehyde fixation is thegeneration of a large number of hydroxymethyl groups through selective interactions withvarious functional groups (e.g., active hydrogens on aromatic rings, primary and second-ary amines, and hydroxyl and sulfhydryl groups) in proteins. The hydroxyl component ofthe hydroxymethyl groups is thought to be reactive, depending on which of these functionalgroups the formaldehyde is bound to. Such an active hydroxyl component could form acoordinated bond with calcium ions (Fig. 6.2). Thus, proteins fixed with the aldehyde maybecome complexed with calcium ions that are abundant in animal tissues, reaching levelson the order of 2 mM in the cytoplasm of eukaryotic cells. These complexes mask epitopesto a variable degree. Calcium complex formation with proteins in this state is likely to bequite strong, involving four to eight coordinate bonds. Therefore, a considerable amountof energy (heat) is required to release the calcium ions from this cagelike complex.

Based on this observation, calcium released from this complex requires high-temperature heating in combination with a calcium chelating and/or precipitating agentsuch as EDTA, EGTA, citrate buffer, or urea. Because these reagents are chelators of divalent

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metal ions, epitope unmasking can be achieved by exposing the sections to such treat-ments. Figure 6.3 shows the disruption of some coordinate bonds at a high temperature inthe presence of EDTA, which results in antigen retrieval. Also, an inorganic salt such assodium carbonate is expected to remove calcium by preferential precipitation. It is inter-esting to note that microwave heating also causes changes in metal ion transport throughthe plasma membrane.

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Further evidence supporting the role of calcium in antigen masking was provided byShi et al. (1999a). They exposed frozen tissue sections to 50mM (pH 7.1) overnightat 4°C and demonstrated a significant loss of immunostaining or altered staining pattern ofantigens such as thrombospondin and Ki-67 compared to controls not exposed toSuch a loss of staining can be partially recovered by incubating the sections in EDTA, sub-stantiating the role of calcium in antigen masking. The antigen masking effect of calciumhas also been demonstrated by adding to antigen retrieval fluid such as EDTA (Kimet al., 1999b). The disodium salt of EDTA (in common use) binds one divalent metal iononly, and the addition of molar excess of calcium ions would eliminate the antigen retrievaleffect of EDTA.

The role of pH in calcium-related effects on antigen unmasking is controversial.According to Morgan et al. (1997b), two different mechanisms are involved in antigenretrieval at acidic and alkaline pH levels. Under acidic conditions (pH 1–3), instead ofchelation, high concentrations of hydrogen ions dissociate calcium complexes and/orbreakdown protein crosslinkages introduced by formaldehyde. On the other hand, in analkaline environment (pH 8.0), chelation of calcium is responsible for antigen retrieval andcan be carried out with a chelator. Dixit et al. (1986) had also proposed earlier that theremoval of calcium by chelation modifies the conformation of protein molecule as anunrolling or unraveling of the large domains, resulting in the exposition of epitopes necessaryfor the binding of certain monoclonal antibodies.

A somewhat different interpretation of the relationship of aldehyde fixation with themasking of antigens with calcium-protein complexes is reported by Shi et al. (1999a).According to this point of view, although calcium-induced modification of the proteinmolecule does occur and can be demonstrated immunohistochemically, it is independentof formalin-induced crosslinking. Addition of calcium chloride can reduce or alterimmunostaining, but it is not related to the pH of this solution.

In conclusion, the effects of calcium bound to tissue are highly complex, for calcium-induced molecular modification may diminish antibody-antigen recognition or enhancethis effect. It is known that the ability of some monoclonal antibodies to recognize theircorresponding epitopes is calcium-dependent under certain conditions. The presence ofcitrate buffer is not necessary to restore antigenicity, provided an appropriate pH is pres-ent. The effect of bound calcium is not the only factor responsible for antigen masking.In addition, modification of a protein molecule also occurs due to crosslinking introducedby formalin fixation, causing antigen masking. Calcium binding to protein moleculesinfluences the immunoreactivity of some epitopes, while others are not affected. On theother hand, heat-induced hydrolysis of protein crosslinks is the primary mechanismresponsible for epitope unmasking. Possible mechanisms responsible for epitope retrievalby heat treatment are summarized below.

In summary, reasoned arguments have been presented in support of several mecha-nisms responsible alone or in combination for antigen retrieval by heating; denaturationand hydrolysis, self-assembly of unfolded protein chains and the subsequent restoration ofantigenic sites, chelation of calcium complexes, and unfolding of protein structure bymetallic salts or urea solutions through dissociation of hydrogen bonds or through the lossof diffusable blocking proteins (Macintyre, 2001). In this respect, the role of residualparaffin in the sections is unclear.

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USE OF ETHYLENEDIAMINETETRAACETIC ACID (EDTA) FORANTIGEN RETRIEVAL

Epitope unmasking can be achieved by exposing sections to high temperature in combi-nation with a calcium-chelating or precipitating agent such as EDTA or EGTA. In fact,maximum antigen retrieval induced by heat is obtained in the presence of EDTA. Such atreatment leads to the extraction of calcium ions tightly complexed to formaldehyde-fixedtissue sections. EDTA solution compared with other antigen retrieval fluids, includingsodium citrate buffer, is more effective in certain cases in augmenting not only stainingintensity but also the number of positively stained cells. It has been reported that EDTAsolution, when combined with heating in a pressure cooker, is more effective than citratebuffer or Tris-HCl buffer in retrieving Ki-67 antigens in gastric and breast cancer tissues(Kim et al., 1999). The intensity of immunostaining of the sections treated with the EDTA-heat combination depends on the pH of EDTA. Strong staining is achieved at pH 3 and atneutral to high pH levels, but the staining intensity decreases at pH 4 and 5.

Another recent study also supports the mediation role of EDTA in unmasking anti-gens (Röcken and Roessner, 1999). In this study thin sections of aldehyde-fixed, Epon-embedded human biopsy tissues were treated with 1 mM EDTA, using a heated water bath.This combined treatment resulted in excellent immunogold staining of amyloid (Fig. 6.4).

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Because heating in water alone did not improve immunostaining, it is concluded that epitoperetrieval is mediated not only by heat and rehydration but also by the presence of a chelatingagent such as EDTA. However, caution is warranted in using EDTA, which may adverselyaffect cell morphology because it is a strong oxidant.

ANTIGEN RETRIEVAL WITH HEAT TREATMENT

Theoretically, any method of heating should unmask epitopes. Although most antigenscan be detected after heat treatment, some may be destroyed, and others may remainmasked. The temperature, changes in temperature during heating, and the duration of heat-ing critically influence antigen retrieval. Different tissues, fixatives, and durations of fixationrequire specific temperatures and durations of heating. Therefore, pilot studies should becarried out to determine optimal heating conditions.

Similar staining intensities are achieved by the following heating conditions irrespec-tive of the heating method used: 100°C for l0min, 90°C for 30min, 80°C for 50 min, and70°C for 10hr (Shi et al., 1995a). Heating at 100°C for l0min (two cycles of 5min each) isrecommended for retrieving most antigens, except those damaged by high temperature. Inthe latter case, lower temperatures for extended durations can be used. Overfixed tissuesrequire high temperatures and/or extended durations of heating. In some cases, durations ofheating longer than 10 min on a full-power setting may cause background staining.

Repeating the boiling cycles is more effective than extending the boiling duration.This can be accomplished by removing the slide jar from the microwave oven after eachrun and placing the slides in a new jar containing the fresh retrieval fluid at room temper-ature, followed by again placing the jar in the oven. Compared with high-power microwaveoutputs, medium wattage (e.g., 450 W) may yield better sensitivity, probably due to optimalthermal effects and hence optimal oscillation of dipolar molecules.

ADVANTAGES OF HEATING

The recognition of many types of antigens by antibodies is facilitated using high-temperatures. Theoretically, high-temperature heating disrupts protein crosslinks introducedby formaldehyde, causes peptide cleavage, and alters protein tertiary structure, resulting inthe exposure of masked epitopes for immunostaining. The retrieval of some types of anti-gens can be accomplished only by heating. Heating allows the use of antibodies thatheretofore could not be employed on sections of tissues fixed with formaldehyde andembedded in paraffin. The heat-induced antigen retrieval procedure lowers the detectionthreshold for the antigen and improves signal-to-noise ratios; this is true for both monoclonaland polyclonal antibodies.

In addition, the development of heating methods permits abandoning the use offrozen tissues, which are difficult to process and study. Another advantage of heating isthat it allows the detection of antigens resistant to proteolytic enzyme digestion andretrieves antigens on sections of tissues left in formalin for prolonged durations.

Generally, heat treatment is superior to enzyme digestion for antigen retrieval.For example, when using polyclonal anti-kappa and anti-lambda antibodies (1:500),

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immunoglobulin light-chain immunostaining was better after microwave heating than aftertrypsin digestion (Ashton-Key et al., 1996). Deparaffmized sections were heated on fullpower in a 750 W microwave oven for 22min, while others were treated with 0.1% trypsinfor 10 min at 37°C. Similarly, in human biopsy tonsil tissue stains better aftermicrowave heating than after trypsin treatment (Fig. 6.5). If the retrieval of an antigen typeis adversely affected by heating, protease digestion of sections is the optimal pretreatment.However, the preservation of cell morphology is generally better in heat-treated than inenzyme-treated sections, especially when extended durations of digestion are employed.

HEATING METHODS

Different heating systems, such as microwave ovens (Shi et al., 1991), pressure cookers(Norton et al., 1994; Miller and Estran, 1995), microwave heating–pressure cookers(Taylor et al., 1995), autoclaves (Bankfalvi et al., 1994), steamers (Taylor et al., 1995),water baths (Kawai et al., 1994), and electric hot plates (von Wasielewski et al., 1994), incombination with antigen epitope retrieval fluids, have been used with various degrees ofsuccess. Although each of the methods has minor advantages and limitations, they yield afairly similar degree of antigen retrieval when appropriate heating conditions are provided.All the processing conditions must be adjusted for a specific study. Such conditions maydiffer from those most widely cited in the literature or recommended by the manufacturers.The choice of the heating method also depends on equipment availability.

A recent comparative study of the following five heating methods using 21 antibodiesalso demonstrated that they produce similar intensities of immunostaining of retrievedantigens provided the heating durations are adjusted appropriately (Taylor et al., 1996b).However, heating methods Nos. 2, 3, and 4 (given below) yield better results. Advantagesand minor limitations of the heating methods are listed below.

1. Microwave heating for 10 min, carried out in a standard, simple, inexpensive, andwidely available microwave oven, is the fastest procedure. Total time required (includingset up of preheating, actual retrieval process, and cool down) is 25 min. A limitation is pos-sible boiling over, resulting in the loss of antigen retrieval fluid. Consequently the level ofthe fluid must be checked every 5 min. If necessary, more fluid can be added after the first5 min to avoid drying the tissue sections. If more fluid is needed, this is the result of boil-ing over, not evaporation. To catch any boiled-over fluid, the slide jar should be placedwithin a larger jar which contains deionized water. In addition, the presence of hot or coldspots in the microwave oven is not uncommon when several isolated jars containing theslides are placed at random in the oven, a practice that leads to reduced reproducibility.This method is also difficult to standardize. The microwave oven (900 W, 2,450 MHz) isset at maximum power for two cycles of 5 min each.

Step-by-Step Protocol

Determine the optimal pH of antigen retrieval solution for each antigen. Citrate buffer(0.01 M) adjusted to pH 6.0 with HC1 is used widely. Determine the desired temperaturebased on the type of tissue and antigen under study. For fatty tissues, 90°C is recommended;adjust the duration of heating accordingly. Place slides in plastic Coplin jars containing the

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Antigen Retrieval 127

antigen retrieval solution in the center of the rotary plate in the microwave oven to ensureuniform heating of slides. Cover the jars with loose-fitting caps. Turn on the microwaveoven and check the temperature of the retrieval solution with a temperature probe. Use amaximum power setting of 7–10.

Start timing the antigen retrieval duration when the retrieval solution begins to boil.As an average, a total retrieval duration of 10 min, divided into two 5-min cycles with an inter-val of 1 min between cycles to check the solution level in the jars, is recommended. If needed,fresh retrieval solution from an adjacent jar can be added to the jars containing the slides.Alternatively, distilled water can be used to replenish the retrieval solution. The objective isto keep the slides fully immersed in the solution before restarting the oven. Alternatively, theduration can be based on previously determined time for sections known to contain the anti-gen under study. Remove jars from the oven, and cool the sections for 20 min at room tem-perature. The slides are ready for immunostaining after being rinsed in 0.5 M PBS (pH 7.4)for 5 min. Do not reuse the antigen retrieval solution. Some of the above-mentioned steps areautomatically controlled by the H2550 Laboratory Microwave Processor.

2. Microwave heating for 20 min is the same as Method 1 except that the heating isemployed four times for 5 min each. Total time required is 35 min. Improved immunos-taining of many types of antigens can be achieved by extending the heating time. The pro-cedure requires attention for 20 min to check the fluid level, and occurrence of hot or coldspots may complicate the procedure.

3. Pressure cooking. Although microwave heating is widely used for antigen retrieval,this system does not raise the temperature of an aqueous buffer above 100°C, even thoughthis temperature is reached rapidly. In contrast, an advantage of the pressure cooker is that,if required, temperatures of 115°C or higher (superheating) can be achieved. Other advan-tages of heating in a pressure cooker include short duration of heating, better repro-ducibility of results with large batches of slides, the ability to use metal slide racks, andeconomy of time and equipment cost (Norton et al., 1994).

Step-by-Step Protocol

Fill to approximately one-third capacity of a domestic pressure cooker (103kPa/15psi) with 0.1 mM citrate buffer (pH 6.0). Bring the buffer to a boil using an electric hotplate, without sealing the lid. Quickly place metal racks containing rehydrated section-mounted glass slides into boiling retrieval buffer, and seal the pressure cooker. Bring thecooker to full pressure. Start timing when the pressure indicator valve reaches the maximum(~4 min). The optimal duration of pressurized boiling is 1–2 min. Depressurize the cookerand cool it under running tap water. Remove the lid, and add cold tap water to replace thehot retrieval buffer. A duration of 15–20 min is required to cool the cooker. Wash the slidesin several changes of 0.05 M PBS (pH 7.4) prior to immunostaining. At no time during thisprocessing are the slides allowed to dry out. The pressurized boiling (120–122°C) longerthan ~2min will progressively degrade the cell morphology.

4. Pressure Cooker–Microwave heatingThe pressure cooker–microwave heating method is simpler than the autoclave proce-

dure and more efficient than microwave heating alone. The pressure cooker does notrequire checking the level of the antigen retrieval solution during heating in the microwaveoven, and a large number of slides can be loaded simultaneously. In addition, the pressure

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cooker does not develop cold spots because it is a larger container. The limitation is that aslightly longer duration (~45 min) is required than that for microwave heating alone.

Step-by-Step Protocol

Place slides in three plastic staining jars, each containing 24 slides and antigen retrievalsolution, and transfer them into a plastic pressure cooker (Nordieware, Minneapolis, MN)filled with 600 ml of distilled water; this amount of water is one-half the capacity of thecooker. Make sure that the jars stand stably in the water (Taylor et al., 1996b). Transferthe pressure cooker into a microwave oven (model R-4A46) which is equipped to switchone power level setting to another automatically. Place the cooker in the center of themicrowave oven. Set the oven at maximum power (900 W, 2,450 MHz) for 15 min to boilthe water, then switched to a 40% power setting for an additional 15 min to maintain mildboiling (simmering). Remove the cooker from the oven, and allow it to cool for 15 min.

5. Autoclave heating, like pressure cooking, provides superheating at temperatureshigher than 100°C. Hydrated autoclaving eliminates the need to adjust the volume of theantigen retrieval solution. Other advantages include the use of larger volumes of theretrieval solution, which gives a uniform heating pattern and allows the heating of a largenumber of slides in a single batch. In this method high-intensity immunostaining isachieved, and the cold spots are absent. Hydrated autoclaving of slides, even in deionizedwater, is thought to be more effective than either microwave or water bath heating (Shin et al.,1991). It is known that heat denaturation of antigens is effective when the protein ishydrated, whereas dehydrated protein is extremely resistant to heat denaturation. Minorlimitations are that the autoclave is expensive and may not be available in some small lab-oratories. Also, the total time required to complete heating is ~45 min. Care should betaken in handling owing to the pressure in the autoclave. The results of autoclave antigenretrieval are shown in Figure 6.6 (Plate 3C, D, E).

Step-by-Step Protocol

Place slides in Coplin jars containing antigen retrieval solution that has been previouslyheated at 80°C. Set the jars in the center of a stainless steel autoclave equipped with a 1,850W heating filament. Tightly close the door of the autoclave as required by the instructions,and heat at 120°C for 10 min at 15 psi. Cool down with running tap water for 20–30 min, thenrinse the sections with 0.05 M PBS at room temperature and immunostain.

6. Steam heating for 20 min over boiling water. Total time required is 35 min. Thismethod has the advantage that loading and unloading of slides into various carriers for auto-mated use is not required. It needs relatively small amounts of the antigen retrieval fluid, doesnot have cold spots, and is inexpensive. This approach is well suited to process a large num-ber of slides simultaneously and thus saves considerable amount of time. Although originallydesigned for autostaining, it can be used manually. A minor limitation is that preheated steamis needed. Slides are set into the TechMate slide holder (Biotek, Santa Barbara, CA), withantigen retrieval fluid in the capillary gap, and are heated by steaming over boiling water.

In contrast to microwave heating, steam treatment heats slides slowly to a uniform tem-perature. This avoids boiling the antigen retrieval fluid and minimizes section detachmentfrom slides. Steam heat used in combination with EDTA and protease digestion has been

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reported to be superior to other antigen retrieval techniques for the immunostaining ofcytokeratin (using antikeratin antibody) in select cases of prostatic carcinoma(Iczkowski et al., 1999). This approach minimizes the diagnostic ambiguity often encoun-tered when using this antibody. Figure 8.8 shows clearly the superiority of the steam-EDTA-protease method over protease treatment alone in detecting acinus with high-gradeprostatic intraepithelial neoplasia. However, for a similar study, the hot plate method ispreferred (see below).

7. Hot plate heating is the simplest and fastest heating method to retrieve antigens insections of formalin-fixed and paraffin-embedded tissues. Certain antigens are optimallyretrieved with the hot plate heating procedure than with enzyme digestion or microwaveheating methods. The hot plate heating method is also most effective in retrieving certainantigens in tissues fixed with formalin for as long as 1 month. The retrieval of basalcell–specific, anti-high-molecular-weight cytokeratin (HMCK) in sections of radicalprostatectomy specimens fixed in formalin and embedded in paraffin has been accom-plished by using the hot plate method; monoclonal antibody clone raised againsthuman stratum corneum was used in this study (Varma et al., 1999).

Step-by-Step Protocol

Sections mounted on a glass slide are placed in a beaker containing 1,000 ml of 0.2M citrate buffer (pH 6.0) and heated on a hot plate (Corning, Utica, NY) for l0min at100°C, and then allowed to cool at room temperature for 20min.

8. Equally good results, if not better in some cases, can be obtained with overnighttreatment of tissue sections in Tris buffer (pH 9.0) in a conventional oven at 70–80°C

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(Koopal et al., 1998). This method has been successfully used for retrieving antigens suchas estrogen and bcl-2 in cervix tissue and lymph node, respectively. Hot oven heating canalso be tried in a humidified chamber.

9. Another antigen retrieval method is hot water bath heating at 90°C for 120min.Total time required is about It is simple and inexpensive, processes a large numberof slides each time, and does not require replenishment of the antigen retrieval fluid. Themethod has been successfully used for the immunostaining of p53 and proliferating cellnuclear antigens (PCNA) (Kawai et al., 1994). For p53 and PCNA retrieval, 0.01 M PBS(pH 7.2) and 0.01 M citrate buffer (pH 6.0), respectively, are recommended (Fig. 6.7). Theuse of this method is limited since it takes much longer time to complete. Hot oven heatingin a humidified chamber can also be tried.

Mechanism of Epitope Retrieval by Microwave Heating

The effects of microwave heating on the tissue sections that result in epitope retrievalare exceedingly complex. A full understanding of the actions of microwaves at the molecularlevel to facilitate epitope retrieval is lacking. At least two mechanisms need to be consi-dered: heat and kinetic energy of the oscillating electromagnetic field. Both possibilitiesare discussed below.

The most commonly accepted point of view is that heat is responsible for unmaskingthe epitopes. In fact, Battifora (1996) has introduced the phrase heat-induced epitoperetrieval (HIER). Heating at 100°C is a powerful treatment that can unmask hidden, buried,or crosslinked epitopes. Heat can be provided not only by a microwave oven, but also byan autoclave, a pressure cooker, steam, or a hot plate. A consensus on which method ofheating is most effective in the retrieval of all types of epitopes is lacking. Therefore, some

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factor or factors in addition to heat also become relevant. It is also known that treatmentsother than heat can also unmask epitopes. Such treatments include enzyme digestion andexposure to detergents.

The aforementioned observations indicate that heat is not the only mechanism respon-sible for epitope retrieval. Therefore, the question arises, is the heat in the microwave oventhe only factor or the primary factor that facilitates epitope retrieval? Is it possible that inaddition to heat, kinetic energy plays a part in epitope unmasking (personal communica-tion, A. S.-Y. Leong)? It is known that the microwave electromagnetic field causes polarmolecules in the tissue to oscillate at a rate of 2.45 billion cycles per second, enough todisrupt protein crosslinking and unmask hidden epitopes. In addition, the fact that ultra-sound treatment, which generates heat for an exceedingly short duration, also unmasksepitopes, suggests that factors other than heat may also be important in explaining thephenomenon of epitope retrieval in a microwave oven. The following explanation mayfurther understanding of the release of thermal energy and heat in a microwave oven.

Microwave energy is a nonionizing radiation (frequency, 300–300,000 MHz) thatcauses molecular motion by migration of ions and rotation of dipoles. Dipole rotationrefers to the alignment, due to the electric field, of molecules that have either permanentor induced dipole moments in both the solvent and specimens. As the field intensitydecreases, thermal disorder is restored, which results in thermal energy being released.At 2,450 MHz (the frequency used in commercial systems), the alignment of the moleculesfollowed by return to disorder occurs times per second, resulting in rapid heating.However, the absorption of microwave energy and its release as heat are strongly depen-dent on the relative dielectric constant (relative permittivity) and the dipolar status of themedium. The relative permittivity is the following ratio: material dielectric constant:vaccum dielectric constant. The greater the relative dielectric constant, the more thermalenergy released, and the more rapid the heating for a given frequency (Camel, 2001).

Due to the particular effects of the microwaves on matter (namely dipole rotation andionic conductance), heating of the section, including its core, occurs instantaneously,resulting in rapid breakdown of protein crosslinkages. Furthermore, the extraction andrecovery of a solute from a solid matrix with microwave heating is routinely obtained inthe field of analytical chemistry (Camel, 2001). However, a definite, full explanation of theeffects of microwave heating on the molecular aspect of antigen retrieval is awaited.

Duration of Microwave Heating

The duration of microwave heating to retrieve epitopes depends on the type of con-centration of the aldehyde used for fixation, duration of fixation, and the temperature inthe microwave oven. The higher the concentration of the fixative and the longer the dura-tion of fixation, the higher the temperature and the longer the duration of microwave heat-ing required for epitope retrieval. The oven temperature is controlled using the temperatureprobe of the oven and is checked with a thermometer. In a microwave oven with 720 Wpower, the boiling point for the epitope retrieval fluid in the Coplin jar isreached in 140–145 sec (Shi et al., 1994). The time it takes to reach a temperature of 55°Cis ~76sec. At 720 W, 5–10 min heating time is recommended, which can be divided intotwo 5-min cycles with an interval of 1 min between cycles to check on the fluid level in

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plastic or glass jars. If necessary, more fluid at the same pH can be added after the first 5 minto avoid drying the tissue sections. The jars can be covered with perforated cling film tominimize evaporation.

Alternatively, 1 hr at 55°C or 30–120 min at 90°C (not boiling) can be used but is notpreferred. With long durations (24 hr–3 years) of fixation with formaldehyde, 20 min expo-sure to heating at 100°C is recommended (Fig. 6.8) (von Wasielewski et al., 1994). A thor-ough washing of slides after microwave heating in the presence of epitope retrieval fluidand before incubation is essential to avoid background staining.

Antigen Retrieval in a High-Pressure Microwave Oven

Although microwave heating, pressure cooking, wet autoclaving, and steaming of tissuesections yield satisfactory antigen retrieval results, comparative studies indicate that pressurecooking or pressure cooking in combination with microwave heating produces more uni-form, efficient, consistent, and rapid immunostaining in some cases (Fig. 6.9). Pressurecooking with or without microwave heating provides temperatures higher than 100°C (super-heating). Such temperatures can be obtained with the high-pressure microwave processorMicroMED URM (Sorisole, B G; Bergamo, Italy) (Suurmeijer and Boon, 1999). This appa-ratus provides controlled superheating under high pressure in the microwave processor.

This processor has a maximal power output of 1,000W. The duration, temperature,and pressure can be adjusted with a touch screen personal computer. Microwave power andpressure are controlled through software. The pressure is regulated as a function of tem-perature, which facilitates heating of the antigen retrieval solution at a constant tempera-ture higher than 100°C without bubbling. A glass dome designed to withstand pressureconditions rotates within the microwave cavity. The dome is provided with an automaticraising and lowering mechanism controlled by the personal computer. A fiberoptic sensormonitors the temperature of the antigen retrieval solution within the dome. The pressure inthe glass dome is between 1,900 and 2,000 mbar.

To obtain antigen retrieval, a plastic jar containing 250ml of 0.01 M citrate buffer(pH 6.0) is centrally placed in the dome within the microwave cavity. Different tempera-tures (ranging from 90–115°C), durations of heating (1–15 min), and pH values (2–10) canbe tested to determine optimal parameters for retrieving a given antigen. For example, opti-mal immunostaining of Ki-67 antigen in malignant tumors using MIB-1 antibody wasachieved at 115°C for 10 min at pH 6.0 (0.01 M sodium citrate buffer) (Suurmeijer andBoon, 1999). To my knowledge the use of this processor has not been reported by anyother laboratory, perhaps because of its high price.

Antigen Retrieval at Low Temperature

Heating treatment is one of the most important factors influencing the effectivenessof antigen retrieval on tissue sections. The heating of sections of the formalin-fixed andparaffin-embedded tissues at a high temperature (boiling) for 10–20 min is extensivelyused for retrieving many types of antigens. A variation of this method consists of heatingat high temperature, followed by heating at moderately low temperature.

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Although antigen retrieval at high temperature yields excellent results in terms ofnuclear immunostaining, cell morphology can be severely damaged. Such damage is eitherignored or not readily visible at the resolution provided by the light microscope. Electronmicroscopy clearly shows this damage. The use of high-temperature heating is especiallyundesirable when studying fatty and fibrous tissues such as breast, skin, and gastrointestinal

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tract; morphological preservation of these tissues can be difficult at high temperatures.Also, sections of these tissues may be dislodged from slides (coated or uncoated) at hightemperature. In some cases, regional staining of the section may also result from high tem-perature heating.

The above-mentioned problems can be generally circumvented by using moderatelylow temperatures (60–80°C) for antigen retrieval. Such temperatures are also useful forantigen retrieval on archival sections that have been stored for years at room temperatureon coated or uncoated slides. Dislodging of archival sections from slides during antigenretrieval is also minimized at moderately low temperatures. According to Biddolph andJones (1999), these problems can be minimized by using low-temperature heating (60°C)in conjunction with boric acid as the antigen retrieval fluid. Loss of archival sections isespecially serious because of the nonavailability of tissue blocks or if all the tissue hasbeen used. If high temperature must be used for archival sections, loss of sections may beminimized by using an adhesive overlay on the sections before heating (Pateraki andKontogeorgos, 1997). This approach requires further testing. Equal staining intensity isusually achieved at 80°C or at higher temperatures. Antigen retrieval can be obtained at 80°Cin 10 mM citrate buffer using a water bath.

A minor disadvantage to using moderately low temperature for antigen retrieval is therequirement of longer heating durations. The lower the temperature, the longer the dura-tion of heating. As an average, heating at 80°C for 2hr is recommended. A wide range ofheating durations at moderate temperature has been used in the published literature, whichare listed below.

Overnight heating (~ 15 hr) at 60°C has been used for retrieving muscle actin (HHF 35)and smooth muscle actin (CCG 7) (Igarashi et al., 1994). The same duration of heating butat 80°C was used in a conventional oven for retrieving estrogen receptor in the cervix tis-sue; Tris buffer (pH 9.0) was used as the antigen retrieval fluid (Koopal et al., 1998).Kawai et al. (1994) have reported that simple heating in a hot water bath at 90°C for 2hrwas very effective for retrieving PCNA and p53 antigens (see Fig. 6.4). In this study,overnight heating at 60°C, using PBS or citrate buffer, also yielded good results. Heatingat 90°C in a microwave oven for 15 min was also used for retrieving myosin heavy chain,using TUF antigen retrieval fluid; boiling was not allowed (Carson et al., 1998). Man andTavassoli (1996) found that overnight heating at 70–80°C produced excellent staining of anumber of antigens, including ER, PR, p53, and Ki-67.

Moderately low temperature in conjunction with enzyme digestion has also beenrecently used for antigen retrieval; a few examples are cited below. The retrieval of Ki-67antigen in surgical breast biopsy specimens has been achieved by treating the sections with0.1% trypsin for 15 min at 37°C, followed by heating in 10 mM citrate buffer (pH 6.0) at80°C for 2 hr in a water bath (Elias et al., 1999). In another study sections of breast cancertissue were pretreated with 0.1% trypsin in PBS (preheated to 37°C) for 15 min, rinsed indeionized water, and then heated in l0 mM citrate buffer (pH 6.0) (preheated) in a waterbath for 2hr for improved retrieval of estrogen receptor and Ki-67 antigen (Frost et al.,2000). A disadvantage of this combined treatment is the focal and sporadic digestedappearance in the sections. These areas can be identified by the presence of inadequatelystained nuclei by hemotoxylin.

Moderate heating with or without enzymatic pretreatment is not the optimal methodof immunodetection of all antigens. For example, more intense staining of antiapoptotic

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protein bcl-2 and progesterone in breast carcinoma is provided by heating in a microwaveoven at full power than by moderate heating, although section loss is not uncommon withfull-power heating. It is deduced from these and other studies that optimal retrieval ofeach antigen or antigen-antibody complex along with the tissue type requires specificintensity of temperature and its duration, and that the source of heat is not very important.Unfortunately, a universal temperature for retrieval of all antigen types in various tissuesremains elusive.

Use of Heat for Staining

Microwave heating accelerates the process of staining for light and electronmicroscopy, although this advantage is more useful for light microscopy because conven-tional staining is quite rapid for electron microscopy. Under microwave heating, thecharged dye ions as well as the polar molecules and ions of the solvent, including water,are excited (Kok and Boon, 1990). The molecular movement generated by microwaveheating may accelerate chemical reactions up to 1,200 times. The result is that heatingspeeds up diffusion of stains into the thick-tissue sections and their subsequent reactionand binding with the substrate.

Generally, compared with conventional staining, staining in the microwave ovenrequires a much shorter staining duration and results in more intense staining, better con-trast, and less nonspecific staining. In fact, the hours required for many conventional stain-ing methods for light microscopy can be shortened into minutes. Several examples arelisted below. One example is the Grimelius method for staining neuroendocrine granulesin various tissues and tumors for light microscopy. The conventional procedure is com-pleted in 3 hr, whereas the microwave method is accomplished in 3 min (Hopwood, 1992).Also, staining of melanin can be carried out with colloidal silver nitrate in 45 sec undermicrowave heating (Leong and Gilham, 1989a). Microwave heating is also effective inreducing the tissue staining time from 70min to 15min for localizing acid and neutralmucins with a modification of alcian blue periodic acid–Schiff stain (Matthews and Kelly,1989). Microwave heat–stimulated staining of the brain tissue with the Rio-Hortego silverimpregnation technique can be completed within 24 hr instead of the 7 days required bythe conventional method (Marani et al., 1987). Satisfactory silver impregnation of cellbodies, axons and their terminals, and dendrites and their spines is obtained.

Another example is the application of the Jones-Marres silver method for rapid stain-ing of fungi in the brain tissue of immunocompromised patients (Boon et al., 1998).This procedure can be carried out using the MicroMED BASIC microwave lab station(Milestone, s.r.l., 24010 Sorisole, Italy). The lab station has software for reliable control ofpower, time, and temperature using infrared temperature control for no-touch temperaturedetermination. It also has a 360-degree rotation carousel (no hot spots) and produces print-outs of the temperature and power levels used during various microwave steps.

Microwave heat can also be applied for rapid staining of frozen sections. Frozen-section diagnosis plays an important role in the evaluation of the operability of the patientand in the examination of resection margins. The preparation for diagnosis, for example ofsignet-ring cell carcinoma in the peritoneum, can be accomplished in as brief a time as 30 secby the modified periodic acid–Schiff’s (PAS) reaction facilitated by microwave heating

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(Dworak and Wittekind, 1992). This protocol can demonstrate even a small number of mucin-containing tumor cells surrounded by fibrous tissue in frozen sections (Fig. 6.10/Plate 3F).

Microwave heat is also effective in staining SDS-polyacrylamide gels withCoomassie blue for visualizing as little as 5 ng of protein against a light blue backgroundof the gel (Wong et al., 2000). This protocol is one of the most powerful methods in mole-cular biology for visualizing proteins.

Rapid staining of frozen sections of human brain tissue that has been stored in 10%formalin (4% formaldehyde) for up to 10 years has also been reported (Feirabend andPloeger, 1991). In this study, rapid staining was obtained in the microwave oven by usingclassic neuroanatomical staining methods such as Klüver-Barrera stain; originally Luxolfast blue step required up to 24 hr, whereas in the microwave oven this step needed only15–60 min. Another application of microwave heating is rapid staining of plant tissues withdyes. For example, Safranin O can stain plant tissues in 45 min at 60°C in a microwaveoven instead of the conventional 48 hr at room temperature (Schichnes et al., 1999).

Microwave heat–assisted rapid fixation and double staining of the mouse fetal skele-ton has also been carried out (Ilgaz et al., 1998). The staining was accomplished with amixture of alcian blue and alizarin red S in 23 min in the microwave oven instead of 4 daysat room temperature. The cartilage and bone are stained distinctly.

Most staining methods require optimal temperatures and durations of staining.The optimal temperatures for most nonmetallic stains is 55–60°C, while for metallic stainsit is 75–80°C (Suurmeijer et al., 1990). Some specific examples are given below: theRomanowsky-Giemsa method and the alcian blue technique at 55°C (Horobin and Boon,1988), the Southgate mucicarmine procedure at 60°C, the Grimelius protocol at 75°C, theGrocott, Jones, and Fontana-Masson methods at 80°C (Kok and Boon, 1990), and the goldchloride (0.02%) at 74–98°C (Noyan et al., 2000). Some dye solutions, such as oil red O,can be used at boiling temperature.

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Table 6.1 indicates microwave power levels, the stain used, and the required temper-ature. Table 6.2 shows significantly reduced duration of staining under microwave heatingcompared with conventional staining conditions. However, many brands of microwaveovens are in use, and all vary in their performance, even at the same power level.Therefore, optimal stain concentration, temperature of staining, and durations of stainingand rinsing will have to be determined for each type of new study. Also, care is requiredin interpreting the staining results because high temperatures tend to produce staining arti-facts. Autostainers for histochemistry are available from the following sources: LeicaAutostainer XL, Leica Instruments GmbH, Nussloch, Germany; Oticmax Rapid MicrowaveHistoprocessor Inc., 160 Shelton Road, Monroe, CT 06468.

Rapid Immunostaining of Frozen Sections

Rapid immunohistochemical study of frozen sections is necessary for intraoperativediagnosis in some cases. Rapid immunostaining is also helpful in confirming or excludingtumor clearance in resection margins or in detecting micrometastases in sentinel lymphnodes in breast cancer patients. Two methods to immunostain frozen sections are theenhanced polymer one-step staining (EPOS) system and the EnVision system; both systemsare detailed later.

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Using the horseradish peroxidase method and microwave heating, Ichihara et al.(1989) were also able to immunostain frozen sections for the intraoperative diagnosis ofpancreatic cancer in 30min. Only four different antibodies were tested in this study.A shorter duration of 10min has also been used for immunostaining frozen sections withthe EPOS system (Chilosi et al., 1994). The EPOS procedure is based on the chemicallinking of primary antibodies and horseradish peroxidase to an inert polymer complex(dextran) (Bisgaad et al., 1993). This methodology has been employed for immunostainingof Ki-67, PCNA, cytokeratin, and leukocyte common antigens (Tsutsumi et al., 1995;Richter et al., 1999). The limitation of the standard EPOS system is that the primary anti-bodies are labeled and thus are commercially available only for limited range of antigens.

In contrast to the EPOS system, a modification of the highly sensitive two-stepirnmunohistochemical EnVision system allows the detection of a broad spectrum of anti-gens in frozen sections in less than 13 min (Kämmerer et al., 2001). In this study 38 out of45 antibodies tested showed specific staining. In fact, the modified EnVision procedureallows the use of any suitable primary antibody, preferably monoclonal antibodies. Likethe EPOS system, EnVision employs a dextran polymer coupled to horseradish peroxidasemolecules for detection. No attempt was made to block endogenous peroxidase, nor wasany antigen retrieval pretreatment used. Because of the very short incubation durations, ahumid chamber is not required to avoid evaporation of immunoreagents.

A minor disadvantage of the modified EnVision system is that it requires primaryantibody concentrations four- to tenfold higher than those used in the conventionalimmunohistochemical procedures. Another limitation of this modified method is that onlytwo slides with two sections each can be processed at any one time.

Enhanced Polymer One-Step Staining Procedure

Sections thick) of freshly frozen tissues are mounted on silane-coated slidesand fixed with 4% buffered formaldehyde (pH 7.0) for 20 sec (Richter et al., 1999). Thesections are rinsed in TBS (pH 7.4) for 15 sec, followed by incubation with EPOS antibodyfor 3 min at 37°C in an incubation chamber. They are rinsed twice for 15 sec each in TBS,and then developed with peroxidase-DAB detection kit (Dako) in a microwave oven (500 W)for ~ 1 min; during microwaving, the slides are cooled by a cold water bath (Werner et al.,1991). After being rinsed in tap water, the sections are counterstained with hematoxylin for10sec. They are rinsed in tap water and cover-slipped.

Modified EnVision Procedure

Tissue specimens are snap-frozen in liquid nitrogen for 30 sec immediately afterremoval and then transferred to a cryostat (Kammerer et al., 2001). Serial frozen sectionsof thickness are cut and placed on silane-coated slides. They are air-dried for 30 sec,fixed in acetone for 1 min at room temperature (22°C), and air-dried at 22°C (Fig. 6.11).The sections are incubated with primary antibody in the antibody diluent (Dako) for 3 minby placing the slide horizontally on a hot plate at 37°C. (All incubation steps are carriedout by placing the slide horizontally on the hot plate at 37°C.) Following a brief rinse inTBS, the sections are incubated with the goat-anti-mouse EnVision-HRP-enzyme conjugatefor 3min at 37°C.

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The sections are rinsed in TBS, and then exposed to DAB+ chromogen (Dako) for afew minutes as the substrate for the EnVision-HRP-enzyme. The sections are washed byshaking the slide rapidly under tap water for 10 sec. The excess fluid is removed from theslide with a paper towel. The slide is dipped in distilled water, counterstained with Meyer’shematoxylin for 15 sec, and then rinsed in hot tap water at 42°C for 30 sec.

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Hazards and Precautions in the Use of Microwave Ovens

The causes of most hazards encountered in using a microwave oven are straightforwardand can be avoided by taking necessary precautions. Higher-power settings and longerdurations of heating than optimal for a given study should be avoided. Because overheat-ing is not uncommon, the time setting should be checked. The fluid contents of the con-tainer heat faster than the container. In fact, the fluid contents of the container heat so fastthat the container can still be cool (Marani, 1998). Even after the container has beenremoved from the oven, it will become hotter for a period of time. Changes in the size,shape, and nature of the container and its position in the microwave oven significantlychange the temperature of the container fluid. Furthermore, changes of these factors willchange the temperature of the container fluid even if the volume of the container contentsremains unchanged.

Overheating the microwave oven tends to result in boiling or excessively rapid evap-oration of fluids such as ethanol used for dehydration, formaldehyde employed for fixa-tion, and the antigen retrieval fluid. As a result, flammable and/or toxic materials arereleased in the microwave oven. Even without overheating, vapors are produced becausecontainers are kept open in the oven to prevent pressurization. Transparent microwave con-tainers should be used, fluid volumes should be ~100ml. Microwave ovens with attachedefficient extractor fans are commercially available, as are microwave ovens with tempera-ture probes. To avoid possible exposure to toxic vapors, the face should be turned awaywhen the oven door is opened (Horobin and Fleming, 1990). The oven door should not beopened or closed to turn the microwave power on and off.

When using Pelco 3440 MAX laboratory microwave oven (Ted Pella, Redding, CA),areas of high microwave flux should be checked, using a Pelco 3,614 microwave bulbarray (Ted Pella) (Fig. 6.12). Specimens should not be placed in areas indicated by illu-minated bulbs. Vials containing the specimens should be placed in a water bath (50 ml) thathas been preheated to the required temperature. The temperature should be regulated byplacing a microwave temperature probe into a vial of the same solution that is present inthe specimen vial. The built-in temperature probe displays the temperature on the ovenfront panel. The wire that attaches the probe to the oven should be submerged in the waterto decrease the antennae effect (Schichnes et al., 1999). An additional 400ml of staticwater load should be placed in the oven at an optimal position determined with themicrowave bulb array. This water is changed between every step.

Pelco BioWave microsystem is the latest advancement in microwave heating tech-nology. It is equipped with vacuum cycling (down to 1 torr), variable wattages, and a pre-cision temperature probe; it can accommodate Pelco coldspot connected to the Pelco loadcooler and thus eliminate hot and cold spots during processing. The system can be used forboth light and electron microscopy. The vacuum chamber is most helpful during fixationand infiltration of tissue specimens.

The following specific steps must be taken while using a microwave oven for antigenretrieval (Marani, 1998).

1.2.3.

Test microwave leakage with a microwave detector with a low sensitivity range.Place the oven in an efficient fumehood.Wear gloves while using your hands inside an oven.

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4.5.6.7.8.9.

Predetermine the maximum power level of the oven to be used.Do not place a container with a closed lid in the oven.Do not use high temperature settings unless absolutely necessary.Predetermine the heating time, and check the time setting.Check the actual temperature attained by the specimen.Predetermine the number of specimens to be heated.

10.11.12.13.

14.15.

Predetermine the exact position of the specimen in the oven during heating.Predetermine the amount of a water load and its place in the oven.Find out the extent of hot spots in the oven.Use Teflon containers with thick walls in the oven. Plastic containers can also beused.Do not use metals or foils in the oven.Contrary to some published reports, pencil-written materials can be used in theoven.

Limitations of Microwave Heating

In spite of the overwhelming advantages of microwave heating, some real and possi-ble limitations are described. Background staining may occur with some antibodies, partic-ularly when the heating is prolonged. This problem can be avoided by determining theoptimal temperature and time of heating by trial and error. Although antigen specificity

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for the monoclonal antibody is maintained after microwave treatment, the possibility ofaltered immunostaining should not be disregarded whenever new or previously untestedantibodies are used. The results of such studies should be compared with those obtainedusing frozen section immunohistochemistry.

In some cases, microwave heating may damage nuclear morphological details,including mitotic figures. This problem may lead to difficulty in identifying cells accu-rately, which is important in diagnostic studies. Both mitotic figures and morphology, forexample, are important in distinguishing a malignant lymphoid infiltrate within a mixedcell population (Hunt et al., 1996). In such studies, it is desirable to use a heating methodother than microwaves and accept slightly lower immunostaining enhancement.

Although the exact knowledge of molecular changes responsible for impairment ofnuclear morphology caused by microwave heating is lacking, it may be possible that thistreatment causes some structural damage to intracellular macromolecules, resulting in anincrease in the number of osmotically active moieties within the nuclear compartment,thus attracting water and causing nuclear swelling (Hunt et al., 1996). Such swelling wouldblur mitotic figures, leading to a less accurate count of them.

Some other limitations and their avoidance are described below. With violent boilingand extensive evaporation of the retrieval fluid in which the sections are immersed, thesections should be monitored to avoid drying and damage. To obviate this problem,microwave heating must be performed in repeated bursts; the plastic jars must be refilledfollowing each cycle or a large reservoir of retrieval fluid or distilled water must be placedin the oven. To avoid inconsistent results, plastic jars containing the slides should alwaysbe placed every time in the same location in the microwave oven. The number of slides andjars should be constant every time a microwave oven is used, even when this entails insert-ing blank slides into the jar (Gown et al., 1993). Tissue sections should be placed towardone end of the slide (lower side of the slide while placing it in the jar) to ensure continu-ous immersion in the epitope retrieval fluid during microwave heating.

Uneven distribution of microwaves within the oven results in hot and cold spots (seepages 102–103). This problem can be avoided by placing a 500-ml water load in the rearof the oven and by using a turntable during the process of heating (Panasonic model NN5652, 800 W). Only a limited number of slides can be accommodated in the microwaveoven. Tissue section detachment from the glass slide may occur during heating, especiallywith tissues containing prominent fibrous elements (Cuevas et al., 1994). If this problemis encountered, the surface of the slide can be made adhesive for sections by coating itwith poly-L-lysine or 3-aminopropyl-triethoxysilane or, still better, by using electricallycharged glass slides. Another problem is that microwave ovens have the inherent disad-vantage of decreased power generation with use. Thus, no two ovens in use will have thesame heating characteristics. This limitation is an obstacle in standardizing antigenunmasking methods.

Microwave heating in some cases is not desirable. This method, for example, causescomplete loss of estrogen receptor immunoreactivity, even when monoclonal antibodyH222 is used (Gown et al., 1993; Leong, 1996). In such cases, alternate procedures, suchas enzyme digestion alone or followed by microwave heating, can be used. Similarly,another steroid hormone receptor androgen shows stronger immunostaining with auto-claving than that using microwave heating (see Fig. 6.13) (Ehara et al., 1996). Anotherexample is insulin, which shows diminished immunoreactivity after microwaving in citrate

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buffer (pH 6.0) (Tornehave et al., 2000). In such cases alternative antigen retrieval fluidsand heating methods are required.

Contrary to some reports, microwave heating in some cases does not abolish con-taminating immunostaining during the consecutive detection of two or more types of anti-gens within the same section. This problem is especially common when doubleimmunolabeling with antibodies of the same species and isotope is used. Recently it wasdemonstrated that microwave heating did not completely abolish contaminating stainingwhen cytoplasmic and nuclear antigens in proliferating cells were labeled in cryostat andparaffin sections, with primary monoclonal antibodies from the same species and the sameisotope being used (Bauer et al., 2001). However, such contaminating staining can beavoided in some cases with the use of microwave heating. Lan et al. (1995) have reportedblocking of antibody cross-reactivity in multiple immunoenzyme staining and retrievingantigens with microwave heating.

In summary, although the microwave heating method is highly effective for detectinga large number of tissue-bound antigens which otherwise may remain masked, primarilydue to fixation with formaldehyde, certain antigens show reduced immunoreactivityfollowing microwave heating. It should also be noted that epitope retrieval withmicrowave heating or other methods can unmask cross-reactivities that can be very diffi-cult to deal with. Therefore, the use of retrieval methods for immunostaining creates thenecessity for increased vigilance in the selection and interpretation of controls, bothnegative and positive.

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WET AUTOCLAVE METHOD

Wet (hydrated) autoclave pretreatment as an alternative to microwave heating wasintroduced by Shin et al. (1991) for unmasking tau protein on sections of brain tissue fixedwith formalin and embedded in paraffin. This methodology was later modified byBànkfalvi et al. (1994) for diagnostic antigen retrieval. The justification for employingautoclaving is that microwave heating is less desirable than equivalent autoclaving for theretrieval of certain antigen types. For example, microwave heating may damage nuclearmorphological details, including mitotic figures. Some evidence indicates that cell mor-phology is preserved better with autoclaving than with microwaving. It has been suggestedthat autoclave heating does not cause loss of sections.

The retrieval of certain types of epitopes requires temperatures higher than 100°C,which is provided by an autoclave (120°C). It is thought that damage by the super-hightemperature to cell morphology is comparatively less in some cases. The super-high tem-perature has been reported to result in stronger immunostaining of steroid hormone recep-tors than with that obtained with microwave heating (100°C for two pulses of 5min each)(Fig. 6.13) (Ehara et al., 1996). Also, compared with microwaving, autoclaving producessuperior immunostaining of progesterone (Mote et al., 1997). In addition, autoclaving yieldsbetter immunostaining of U2-OS cell nuclei for retinoblastoma susceptibility gene product(RB) compared with that achieved by using microwave heating (Tsuji et al., 1998).Furthermore, immunostaining of fibronectin epithelial nucleus in oral mucosa was not presenton frozen sections, but such staining was achieved after autoclaving (Mighell et al., 1995).

However in some cases, autoclaving is undesirable. This is exemplified by the aboli-tion of immunostaining of calcineurin in the CNS neurons after autoclaving (Usuda et al.,1996). Autoclave pretreatment (100°C) may damage cell morphology of fatty tissues ortissues containing areas of fatty tissues (e.g., breast tissue). The extent of damage can beminimized by mounting the sections on protein-coated glass slides that have been allowedto dry for 48–72 hr before autoclaving (personal communication, 1999, K. W. Schmid).

In addition to its effectiveness in antigen retrieval in the cases above, an autoclave hasthe advantage of accommodating a much larger number of slides than does a microwaveoven. Several hundred slides can be simultaneously processed in an autoclave, eliminatingpossible immunostaining variations when small batches of slides are microwave-heated atdifferent times. Moreover, antigen retrieval fluid is not lost during heating in an autoclave.In contrast, in a microwave oven the jar containing the slides must be refilled after eachheating cycle. However, this tedious exercise can be avoided by using a large reservoir offluid to minimize the possible deleterious effects of boiling or drying of the sections.A drawback of autoclaving is the high cost of the equipment. The use of a pressure cookeris a cheaper alternative for a smaller number of sections.

Procedure 1

Tissues are fixed with formalin for 18 hr to 4 weeks and then embedded in paraffin.Sections are mounted onto superfrost or poly-L-lysine–coated glass slides, driedin an oven for 1 hr at 60°C, and deparaffinized with three changes of xylene. This is followedby rehydration through a series of descending concentrations of ethanol. The slides are placed

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in plastic Coplin jars containing 0.01 M sodium citrate buffer (pH 6.0), which are heated in anautoclave for 5–10 min at 120°C. The slides are allowed to cool down to room temperature for20–30 min and then briefly rinsed in 0.05 M Tris-HCl buffer (pH 7.4) or 0.1 M PBS.

Blocking of endogenous peroxidase activity is accomplished by immersing the sec-tions for 30 min in a solution of 0.3% in distilled water, and then rinsing in PBS. Ifneeded, background staining can be blocked by treating the sections for 10–30 min at roomtemperature with normal serum from the species supplying the second antibody at a dilu-tion of 1:5 to 1:20 in PBS. The sections are incubated overnight in a humidified chamberat 4°C in the primary antibody at an appropriate dilution. They are rinsed three times for5 min each in PBS, further processed by using avidin-biotin complex (Vectastin, VectorLabs, Burlingame, CA), followed by DAB as the chromogen. Counterstaining of the nucleiis accomplished with hematoxylin or methyl green. As a negative control, irrelevant anti-body UPC10 (Cappell, Organon Teknika, West Chester, PA) can be used or primaryantibody can be omitted. The sections are mounted in an appropriate mountant.

Procedure 2

The following hydrated autoclave method can be employed for immunohistochemicaldetection of molecules in both cultured cell and tissue specimens. The method was used, forexample, to localize androgen receptor in cultured LNCaP cells (derived from prostaticcarcinoma metastasized to lymph node) and biopsy specimens from patients with prostaticcarcinoma (Ehara et al., 1996). After being removed from the culture medium, the cells onplastic cover slips are fixed with 10% formalin for 10 min at 20°C. Tissue specimens are fixedfor 1–2 days and embedded in paraffin. Sections are cut, mounted on glass slides, andheated in an oven for 1 hr at 42°C to promote adherence to the slide. After deparaffinizingand rehydration, the sections are subjected to epitope retrieval treatment as follows.

The slides are placed in metal slide racks and immersed in a beaker filled with 0.01 Mcitrate buffer (pH 6.0). The beaker is loosely covered with a sheet of aluminum foil andautoclaved for 15 min at 120°C. After cooling to room temperature, the autoclave lid istaken off. The sections are treated with 3% hydrogen peroxide in methanol for 15 min toblock endogenous peroxidase activity. As a blocking solution, 10% normal goat serum isused for 10 min. The sections are reacted with the primary antibody at an appropriate dilu-tion at 4°C in a moist chamber. After being washed with 0.075% Brij 35 (Sigma ChemicalCo., St. Louis, MO) in PBS three times, the sections are treated with an appropriately predi-luted antibody for ~10 min in a moist chamber. After washing, the sections are reacted withthe prediluted HRP-labeled streptavidin for ~5 min. The sections are washed, and the HRPsite is visualized with DAB, hydrogen peroxide, cobalt, and nickel, without counterstaining.As a negative control, the sections are reacted with normal mouse serum, normal IgG, ornormal rabbit serum in place of the specific antibodies after autoclaving (see Fig. 6.13).

ULTRASOUND TREATMENT

Ultrasound (sonication) converts AC line voltage to 20-kHz high-frequency electricalenergy, which is fed to a converter where, in turn, it is converted to mechanical vibrations.

Antigen Retrieval 147

The main part of the converter is a lead zirconate titanate electrostrictive element thatexpands and contracts when subjected to alternating voltage (Portiansky and Gimeno,1996). The converter vibrates in a longitudinal direction and conveys this motion to thehorn tip immersed in the solution, resulting in the implosion of microscopic cavities in thesolution. The implosion causes the molecules in the solution to become exceedinglyagitated. This phenomenon is explained below.

Some information is available on the mechanisms responsible for the direct or indirecteffects exerted by ultrasound on antigen retrieval. Considerable heat is generated duringultrasound exposure, but the heat dissipates very quickly. Very rapid heat loss has misledsome workers to state that “ultrasound generates a mild increment in temperature”(Portiansky and Gimeno, 1996).

Ultrasound waves consist of cycles of compression and expansion. Compressioncycles exert a positive pressure on the liquid, pushing the molecules together, whereasexpansion cycles exert a negative pressure, pulling the molecules away from each other.The tensile strength of solutions is reduced by gas trapped in the crevices of small solidparticles in the solution. When a gas-filled crevice is exposed to a negative pressure cyclefrom a sound wave, the reduced pressure makes the gas in the crevice expand until a smallbubble is released into the solution, initiating cavitation. A negative pressure of only a fewatmospheres will form bubbles. The bubbles ( in diameter) implode violently inless than a microsecond, intensely heating their contents (Suslick, 1989). Thus, during theexpansion cycle a sound wave of sufficient intensity can generate cavities in the solution.

Ultrasound treatment causes enormous molecular agitation (turbulence), heat, andpressure of imploding cavities. Such agitation not only initiates but also accelerates bothbiochemical and physical reactions. In other words, effects of ultrasound involve processesthat create, enlarge, and implode gaseous and vaporous cavities in a solution. The implo-sion of cavities also sends shock waves through the solution. This extreme condition gen-erated by cavitation can induce reactivity between cellular proteins and the antigenretrieval solution (e.g., sodium citrate). Mechanical vibrations and high temperatures mayextract tissue-bound calcium ions, accelerating the chelating effect of citrate. This sugges-tion is reinforced by the evidence that ultrasound hastens calcium chelation and bonedecalcification (Thorpe et al., 1972; Page et al., 1990). Chelation of calcium may result inepitope retrieval (Morgan et al., 1994).

It is known that ultrasound can break or disrupt cells and tissues. Mechanical vibra-tions generated by ultrasound can induce structural changes in the tissue sections, break-ing the formalin-introduced protein crosslinks and thus facilitating the accessibility ofantigens to antibodies. Ultrasound can also unfold or “crack” protein molecules intosmaller fragments, exposing the epitopes.

The effectiveness of ultrasound treatment in epitope retrieval has been compared withthat achieved with microwave heating or pressure cooker alone (Portiansky and Gimeno,1996). It was shown that ultrasound was more effective in immunostaining prostatic basalcell structural cytokeratins. The capability of microwave heating for epitope retrieval has alsobeen compared with that of ultrasound in combination with microwave heating (Brynes etal., 1997). The latter approach resulted in stronger immunostaining with lower nonspecificbackground staining of cyclin Dl bcl-1 nucleoprotein in mantle cell lymphoma specimens.It should be noted that raising the temperature beyond a certain level in the presence orabsence of ultrasound does not improve epitope retrieval and in addition results in excessive

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background staining. A limitation of ultrasound heating is that it is difficult to reproducebetween laboratories, as the exact parameters regarding the intensity at the acousticfrequency are difficult to set precisely.

The results of the studies carried out by Hammoud and Van Noorden (2000) usingultrasonication do not agree with those reported by Portiansky and Gimeno (1996). Theformer authors do not recommend using this technique in a routine histopathology labora-tory. Miller et al. (2000) also report that the results obtained with ultrasonic treatment areinferior to those achieved with a pressure cooker. The discrepancy between the resultsobtained with these two teams and other workers may be due to the difference in the typeof antigens ultrasonicated in various laboratories under different processing conditions.For example, Portiansky and Gimeno (1996) used an ultrasonic cell disrupter with an out-put of 40 W, whereas Hammoud and Van Noorden (2000) employed an ultrasonic clean-ing bath having an output of 80 W. Also, various studies used tissues fixed for differentdurations. Tissue section adhesion to slides has also been reported to be a problem duringultrasonication. In spite of the lack of agreement on the usefulness of ultrasonic treatment,this method requires very short duration (~40 sec) for antigen retrieval. However, the use-fulness of ultrasonic treatment requires additional substantiation.

Procedure

Tissues are fixed with 10% formalin for 7–10 days and embedded in paraffin(Portiansky and Gimeno, 1996). Sections about thick are mounted on glass slidescoated with poly-L-lysine and deparaffinized with xylene. They are incubated with 0.03%methanolic hydrogen peroxide for 30 min to inhibit endogenous peroxidase activity.Following dehydration with graded ethanol, they are rinsed in deionized water and then inPBS. The glass slides containing these sections are vertically oriented in the lateral walls ofa 75×95-mm glass dish and completely covered with 10 mM citrate buffer (pH 6). The tipof the cell is disrupted (Branson Ultrasonics model 250), set to continuous mode, andimmersed 3 cm in the citrate buffer in the center of the dish. After incubation in the primaryantibody (appropriately diluted), the avidin-biotin complex (ABC) is used as the detectionsystem. In control sections, the primary antibody is replaced with normal mouse serum.

NONHEATING METHODS

Detergents

Antigen retrieval using heat-based methods is not being widely used for cell culturesand cryosections fixed with an aldehyde. The immunolabeling efficiency of such specimenscan be improved by using a chemical antigen retrieval protocol. This protocol consists of per-meabilizing the specimens with Triton X-100, followed by treating with sodium dodecylsulfate (SDS). This permeabilization/denaturation treatment is applied after fixation and priorto incubation with the primary antibody. SDS is the most commonly used denaturing agentfor gel electrophoresis. The application of SDS in epitope retrieval is based on the observa-tion that after treatment with this reagent, protein bands appear in gel electrophoresis, but

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such results with similar proteins without SDS treatment using immunocytochemistry arenot visible. Being a protein denaturant, SDS application may result in bands after stainingwith Coomassie blue, but it itself is not a stain. Therefore it should not be referred to as apositive or negative stain.

SDS disrupts noncovalent interactions between subunits of a protein, so if a proteinhas two subunits, two bands will appear. In the absence of SDS, only one band will appear.This reagent and mercaptoethanol reduce protein subunits that are disulfide bonded. Thisproperty of SDS may be responsible for protein denaturation. It should be noted that SDSalso permeabilizes cells for antibody access to intracellular epitopes.

Triton X-100 and digitonin are also used to permeabilize the cell membrane allowingantibody penetration. Triton X-100 permeabilization of formaldehyde-fixed cells allowsantibodies better access to their epitopes than does digitonin treatment. Digitonin or saponinbinds to cholesterol within membranes, creating digitonin-cholesterol complexes and poresin the membrane. The pores are sufficiently large to allow antibody penetration. On theother hand, Triton X-100 is a stronger detergent and dissolves most of the membrane lipids.As a result this detergent increases the accessibility of antibodies to cell compartments thatare not permeabilized with digitonin (Hannah et al., 1998). A limitation of Triton X-100 isthat it may extract certain antigens even from fixed cells. Thus, false-negative staining ofantigens, especially membrane antigens, of the cells treated with a strong detergent canoccur because of antigen extraction. However, it should be noted that not all membranes offormaldehyde-fixed cells are impermeable to antibodies without permeabilization.

The permeabilization/denaturation method has been successfully used for immunola-beling of in human neutrophils and MRC-5 cells (Robinson and Vandré, 2001).The method has also been effective in labeling MDCK cells in conjunction with indirectimmunofluorescence (Brown et al., 1996). It should be noted, however, that some type ofantigens remain masked, while other types may be adversely affected by SDS treatment.Still other antigens (e.g., aquaporins and brush border gp330) remain unaffected by SDStreatment (Brown et al., 1996). An example of an antigen whose staining is negativelyaffected by SDS treatment is in the Golgi complex; this occurs with the anti-AE1 anionexchanger antibody (Brown et al., 1996). Therefore, the usefulness of the SDS treatmentshould be assessed in each case. Caution is also required to prevent drying out of the speci-mens during incubation steps because they become hydrophobic with SDS treatment.

Procedures

Kidney tissue is fixed with paraformaldehyde-lysine-periodate by vascular perfusion(Brown et al., 1996). Tissue slices are further fixed overnight at 4°C with the same fixativeand stored in PBS (pH 7.4) containing 0.02% sodium azide. They are placed in 30%sucrose in PBS for at least 1 hr, and then surrounded by a drop of Tissue-Tek embeddingmedium on a cryostat chuck before freezing by immersion in liquid nitrogen. Cryostat sec-tions about thick are cut at a chamber temperature of –25°C, collected on FisherSuperfrost Plus charged slides, and stored at –20°C until use.

The sections are brought to room temperature, and a wax pen (PAP pen, KiyotaInternational) is used to trace a hydrophobic circle around each section. They are rehy-drated by immersion in PBS for 5 min; most of the PBS is removed from the slide with atissue paper and the sections are then covered with drops of SDS solution (1% SDS in

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PBS). The drops are confined to areas where the sections are encompassed by the wax cir-cles. After the slides have been treated horizontally for 5 min at room temperature, the slideis immersed in PBS in a Coplin jar to remove the SDS. The control slide not exposed toSDS is washed in a separate jar to avoid any contact with SDS. The slides are thoroughlywashed three times for 5 min each with PBS, completely removing the SDS; otherwise,residual SDS will denature the antibodies subsequently applied to the sections.

While the slide is horizontal, excess PBS from the areas outside the wax circles isremoved. The sections become hydrophobic after SDS treatment, so care must be taken toprevent them from drying. The aliquot of primary antibody should be already in thepipette, so that it can be applied to the sections immediately after the residual PBS hasbeen removed. The sections can be incubated in the primary antibody for 1–2 hr at roomtemperature, followed by two washes for 5 min each in high-salt PBS (containing 2.7%NaCl instead of 0.9% NaCl). This PBS minimizes nonspecific binding of antibodies to thetissue. After being washed for 5 min in normal PBS, the sections are incubated in the sec-ondary antibody (goat antirabbit IgG conjugated to fluorescein isothiocyanate, FITC) for1 hr. This is followed by washing in normal PBS, then mounting of sections in the mediumof choice (Fig. 6.14).

A second nonheating epitope retrieval method involves the use of sodium hydroxide-methanol solution. This solution was used successfully for epitope retrieval in sections offormalin-fixed, acid-decalcified human temporal bone embedded in celloidin (Shi et al.,1991). This solution is prepared by adding 50–100 g of NaOH to 500 ml of methanol in abrown bottle and mixing vigorously. The solution can be stored for 1–2 weeks at roomtemperature; it is also available commercially (BioGenex, San Ramon, CA). The clear,saturated solution is diluted 1:3 with methanol before use. A wider application of thissolution is awaited.

Another reagent used to unmask epitopes by denaturing antigens is guanidinehydrochloride (GdnHCl) which is freely soluble in water and alcohol; its

Antigen Retrieval 151

aqueous solution has neutral pH. It was used for retrieving masked or hidden intracellularprotein in scrapie-infected cultured cells (Taraboulus et al., 1990). A modified version ofthis protocol was employed for localizing different epitopes in BHK-21 cells fixed withparaformaldehyde containing small amounts of glutaraldehyde (Peränen et al., 1993).These epitopes were undetectable without denaturing the antigens with GdnHCl, usingimmunofluorescence microscopy.

The advantage of denaturation of antigen complexes using GgnHCl is that it allowsthe use of glutaraldehyde, a more potent protein cross-linker, which better preserves pro-tein cell structure. This means that low concentrations of this dialdehyde do not interferewith the epitope retrieval property of GgnHCl. The use of glutaraldehyde widens the appli-cations of this denaturing agent. Both monoclonal and polyclonal antibodies can be usedin conjunction with GgnHCl. Guanidine hydrochloride is especially useful with antibodiesknown to react only with denatured antigens. This reagent also permeabilizes cells.

A limitation is that GgnHCl tends to eliminate the antigenicity of certain intra-cellular structures such as microtubules. However, microtubule loss can be preventedby using low concentrations of glutaraldehyde during fixation with paraformaldehyde(Peränen et al., 1993).

Proteolytic Enzyme Digestion

A variety of proteolytic predigestions have been employed for unmasking epitopes thathad become inaccessible as a result of crosslinking during aldehyde fixation. The digestivetreatments have been carried out most commonly with trypsin, pepsin, proteinase K,or pronase (their concentrations are given later) prior to immunostaining. Detailed com-parative studies on the effects of these four enzymes on epitope unmasking demonstratethat while the results did not differ significantly among themselves, their effects did differ,depending on the tissue and the antibody used (Hazelbag et al., 1995). Other factors affect-ing such results include the duration of digestion, pH, temperature, and length of fixation.

The mechanism responsible for antigen retrieval by enzymatic digestion is break-down of protein crosslinks formed during formalin fixation. It is likely that enzyme treat-ment digests surface binding proteins, exposing the masked antigenic sites for antibodybinding. This idea is supported by evidence that the duration of enzymatic digestionrequired for epitope retrieval is proportional to the length of formaldehyde fixation. It isalso known that overdigestion leads to damage, not only to cell morphology but also toimmunoreactivity.

Enzymatic digestion is preferred over microwave heating for antigen retrieval in a fewcases. Even multiple enzymatic digestion is required to retrieve certain antigens in a spe-cific tissue. As an example, it has been reported that the monoclonal antibody RCC is mosteffective in the staining of clear cell carcinomas and papillary carcinomas in renal neo-plasms when sections are pretreated with a three-step enzymatic digestion method: 0.12%trypsin in Tris-buffered saline (TBS), 0.01% pronase in TBS, and 0.1% pepsin in 0.1 NHC1. Results were inconsistent with heat-induced epitope retrieval techniques. However,trypsin is used most commonly, which catalyzes the hydrolysis of orginyl and lysyl pep-tide bonds. Trypsin usually is used at a concentration of 0.1% in 0.05 M Tris/HCl buffer(pH 7.8) containing 0.1% for 20–40min at 37°C. The addition of is essential

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for controlling digestion and reducing the production of occasional white flocculation(Macintyre, 2001). Only freshly prepared solutions of these enzymes should be used, asenzyme activity decreases with age. Also, these solutions should be prewarmed to therequired temperature to ensure consistent results.

Proteolysis does have certain limitations. Some antigens are susceptible to enzymedigestion. In some cases insufficient unmasking can result in poor or false-negative results,while excessive digestion may adversely affect cytomorphological features and causeincreased background staining and detachment of tissue sections from the slide. It isknown that proteolysis is a potent treatment. Because the cleavage of the protein moleculeby proteolytic enzymes is mostly nonspecific, these reagents may alter the epitopes.In other words, peptide bond cleavage by these treatments is largely nonspecific. Therefore,these procedures are not the preferred treatments. Nevertheless, enzymatic digestion isuseful for a limited panel of antibodies. If needed, enzymatic pretreatments can be appliedpreceded by microwave heating (Dookhan et al., 1993).

Procedure

Slides with tissue sections are treated with 0.1% trypsin solution containing 0.1%(pH 7.4) for 15 min at 37°C, with 0.4% pepsin solution containing 0.01 MHC1 for

20 min at 37°C, or with 0.025% pronase E solution containing 0.05 M Tris-HCl (pH 7.6)for 15 min at the same temperature (Hazelbag et al., 1995). These are average concentra-tions and durations, which should be adjusted according to the tissue and antigen type andthe duration of fixation. Prolonged fixation requires longer proteolysis to unmask the epi-topes. Excessive proteolysis results in decreased immunostaining. If loss of the sectionsduring proteolysis is a problem, the slide can be coated with a 3% solution of casein whiteglue and dried overnight before the sections are placed on it.

Enzyme Digestion and Relatively Low Temperature (80°C)–AssistedAntigen Retrieval

High-temperature microwave heating is currently the most widely used antigenretrieval method. This approach has significantly improved the detection of a wide varietyof antigens. In some studies low-temperature (80°C) antigen retrieval is more effectivethan that obtained with high temperature. This is exemplified by restoration of estrogenand progesterone immunoreactivity (Elias and Margiotta, 1997). Recently, it was reportedthat sequential use of trypsin digestion and low-temperature heating (80°C) was moreeffective than high-temperature retrieval of Ki-67 antigen in breast tumors, using MIB-1antibody (Elias et al., 1999). Another reported advantage of the former approach is that itcauses the least amount of section loss during heating; sections of tissues with a high fatcontent may be dislodged from the slide at a high temperature.

Surgical breast biopsy specimens are first fixed with neutral buffered formalin (4%)for 4–6 hr, followed by zinc-formalin for 2 hr. Paraffin sections ( thick) are placedon silane-coated slides, dried on a slide warmer (60°C) for 1 hr and then in an oven (60°C)for an additional 1 hr. Deparaffinized sections are digested with 0.1% trypsin in PBS at 37°Cfor 15 min. The sections are placed in 10 mM citrate buffer (pH 6.0) and transferred into awater bath (80° or 90°C) for 2 hr. After a 20-min cooling period, the sections are rinsed

Antigen Retrieval 153

with PBS and then incubated overnight at 4°C in the MIB-I antibody (Immunotech,Westbrook, ME), diluted 1:50 with PBS.

An automatic immunostainer (Cadenza, Shandon Scientific Inc., Pittsburgh, PA) canbe used to accomplish staining. The Supersensitive Streptavidin-AP detection kit(BioGenex, San Ramon, CA) is used according to manufacturer’s directions. The finalcolor reaction is developed with a fast red substrate (BioGenex), followed by mild hema-toxylin counterstaining to avoid masking weak immunostained nuclei. Slides are cover-slipped with Crystal Mount (Biomedia Corporation, Foster City, CA).

COMPARISON OF ANTIGEN RETRIEVAL METHODS: A SUMMARY

The following recent comparative studies demonstrate that no single antigen retrievalmethod is optimal for all types of antigens.

1.

2.

3.

4.

5.

6.

7.

8.

9.

Immunostaining of and isoforms of calcineurin in the human brainemploying CAN-2 and CAN-3, respectively, was compared between microwaveheating (in 10 mM sodium citrate at pH 6.0 for 10 min) and autoclaving at 120°Cfor 20 min (Usuda et al., 1996). The former approach was the most effective forintensification of the immunoreaction.Compared to enzyme digestion methods, microwave heating demonstrated moreintense immunoreactivity of estrogen and progesterone in breast cancer tissuesfixed with methacarn (60% methanol, 30% chloroform, and 10% acetic acid)(Oyaizu et al., 1996).Compared with trypsin digestion, microwave heating produced more consistentresults and was effective over a greater range of fixation tissues in the case ofimmunoglobulin light chain in tonsil tissue (Ashton-Key et al., 1996).Compared with pepsin predigestion, microwave heating markedly enhanced thestaining of aberrant p53 antigen with Pab 1801-D07 antibody cocktail in paraffinor frozen sections in adenocarcinoma of the lung (Resnick et al., 1995).Compared with microwave heating (three times for 5 min each at 100°C), hydratedautoclaving (5 min at 121°C) yielded stronger immunostaining of bcl-2 using bcl-2,124 antibody (Umemura et al., 1995).Compared with nonhydrated autoclaving, hydrated autoclaving produced strongerimmunostaining of tau (a microtubule-associated protein) using anti-PHF/tau andantihuman tau (Shin et al., 1991).Compared with microwave heating, heating on a hot plate yielded better immunos-taining of IgG using antihuman IgG on epoxy thin sections for electronmicroscopy (Stirling and Graff, 1995).Compared to microwave heating, superheating (120–122°C) for 1–2 minutes in apressure cooker gave better immunostaining of IgD (Norton et al., 1994).Immunostaining of a wide variety of biopsies was studied using different antigenretrieval fluids and heating and digesting systems (Pileri et al., 1997). This studyshowed the superiority of pressure cooking and EDTA over other methods, includingmicrowave heating and proteolytic treatment.

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10.

11.

12.

13.

14.

15.

16.

17.

18.

Ultrasound treatment was compared with microwave heating and pressure cooking;the former treatment was claimed to be quantitatively and statistically superiorfor the immunostaining of prostatic basal cell structural cytokeratins using mon-oclonal antibody K8.12 (Portiansky and Gimeno, 1996).Compared with trypsinization–microwave heating, microwave heating–trypsinization demonstrated optimal immunostaining of Ki-67 using monoclonalantibody MIB-1 (Szekeres et al., 1995).Immunostaining using a panel of 21 antibodies was compared by employingmicrowave heating, microwave–pressure cooking, autoclave, and steamer (Tayloret al., 1996b). These methods yield similar intensities of staining provided thedurations of heating are appropriately adjusted.Immunostaining of cytokeratin 18 in normal and neoplastic hepatocytes usingantibody CK 18 was compared by employing microwave heating (15 min), auto-claving (10 min), pressurized boiling (1min), and simple boiling (15 min) in10 mM citrate buffer (pH 6.0) (Xiao et al., 1996). No difference was found in thedegree of immunostaining with light and electron microscopy.Compared to microwave heating, digestion with proteinase K for 2–4 min atroom temperature yielded better retrieval of cytokeratins in mouse tissues usingmonoclonal antibodies (e.g., AE1, AE3) generated against human cytokeratins(Martin et al., 2001).Immunostaining of a number of proteins between microwave heating at 100°C of20 min and boiling on a conventional hot plate. No difference was observed in theresults of the two methods (Varma et al., 1999).Antigens bcl-2, CD3, and CD79a in tonsil tissue embedded in methyl methacrylateshow superior immunostaining with trypsin followed by superheating at 121°C ina pressure cooker compared with that obtained with microwave heating only(Hand and Church 1998).Among the three antigen retrieval methods, hydrated autoclaving, microwaveheating, and simple heating, simple heating overnight at 60°C was most effectivefor smooth muscle actin labeling (Igarashi et al., 1994).More intense and widely distributed staining of cytokeratins was observed withprotease digestion than with microwave heating in benign lesions in the prostateusing mouse monoclonal antibody (Googe et al., 1997).

Chapter 7

Antigen Retrieval onResin Sections

Most commonly, antigen retrieval involves heating sections of paraffin-embedded tissuesprior to light microscopy. However, antigen retrieval can also be accomplished on sectionsof resin-embedded tissues (Fig. 7.1). Tissues embedded in a resin show superior preservationof cellular details compared with those embedded in paraffin. Moreover, resin sections per-mit high resolutions to be obtained. In addition, semithin or thin (8–100 nm)sections can be obtained from resin-embedded tissues, allowing correlative studies usinglight and electron microscope, respectively (see Fig. 1.3). For details of resin microscopy andon-section immunocytochemistry, the reader is referred to Newman and Hobot (2001).

The immunostaining quality of resin sections is usually comparable to that yielded byparaffin sections. Prior microwave heating of resin sections results in enhanced immunoreac-tivity with specific, easily interpretable staining using a variety of antibodies. In addition, resinsections allow immunogold and immunogold-silver immunostaining. Excess backgroundstaining is not a problem with resin sections, provided they are premicrowaved or heated byother means. In some cases, resin sections may show less intense staining than that exhibitedby paraffin sections, which is due to thinness (~80 nm) of the former sections. Also, positivestaining may not be achieved in some cases. This is exemplified by antibodies to neutrophilelastase and CD61, which show negative immunostaining on resin sections even aftermicrowave heating (McCluggage et al., 1995). In contrast, immunostaining of CD20 is morereliable on resin sections than on paraffin sections of bone marrow trephine biopsy specimens.Note that the reaction of antibody with antigen is a surface phenomenon in resin sections.

Various types of resins can be used for tissue embedding for antigen retrieval. Bothwater-miscible and water-immiscible resins (Hayat, 2000a) can be used in immunostain-ing for light and electron microscopy. Water-miscible resins used in light microscopyinclude the acrylic polymer glycol methacrylate (Suurmeijer and Boon, 1993b) and LRWhite (Sormunen and Leong, 1998), as well as the hydrophobic resins methyl methacrylate(Hand et al., 1996) and Polybed 812 (McCluggage et al., 1998). All these were used withprior microwave heating. Recently, using EDTA and heat, antigen retrieval was accom-plished on Epon sections (Röcken and Roessner, 1999).

The following embedding mixture is excellent when sections of thickness arerequired. It has been employed for embedding bone marrow trephine biopsy specimens for

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light microscopy (McCluggage et al., 1995):

Polarbed 812 50 gDDSA 32 gMNA 21 gDMP-30 2 g

DDSA: dodecenylsuccinic anhydrideMNA: methyl nadic anhydrideDMP-30: 2, 4, 6, tris (dimethylaminomethyl) phenol

ROLE OF FIXATIVE AND EMBEDDING RESIN INANTIGEN RETRIEVAL

It is well established that formaldehyde reacts with amino groups on protein side chains(Fig. 7.2), introducing mostly reversible protein crosslinks. Epoxy monomer reacts with the

Antigen Retrieval on Resin Sections 157

new hydroxyl groups introduced on the protein by formaldehyde; the epoxy moleculesthereby are copolymerized with the protein. In other words, formaldehyde functions as a linkbetween protein side groups and the epoxy monomer (Brorson et al., 1999). In contrast, sucha copolymerization does not occur between acrylic resins and tissue proteins. These resinspermeate the tissue without chemically binding to them. Accordingly, during thin section-ing, the two resins cleave differently. In the case of acrylic resins, the surface of cleavagetends to follow the path of least resistance; this path is the interface between the resin andproteins. Thus, more epitopes without splitting are exposed at the surface of acrylic sections.

On the other hand, in the case of epoxy sections, the resistance in such interfaces isnot significantly less than that in tissue proteins, which results in the splitting of protein

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molecules. Also, the surface of epoxy sections is smoother than that of acrylic sections.Consequently, fewer epitopes are exposed on the surface of the former sections. However,epoxy resins are of value, being easier to cut and more stable under the electron beam andbetter preserving the ultrastructure.

IMMUNOSTAINING OF THIN RESIN SECTIONS

Antigen sites can be unmasked not only on thick and semithin resin sections for lightmicroscopy but also on thin resin sections for electron microscopy. Antigen retrieval at theultrastructural level has been accomplished on thin sections of epoxy resins (Stirling andGraff, 1995; Röcken and Roessner, 1999) and LR White resin (Wilson et al., 1996;Sormunen and Leong, 1998). Because epoxy and LR White resins are superior to someother resins with respect to preserving the cellular details and other characteristics, antigenretrieval methods using these two resins for electron microscopy are presented.

For electron microscopy, tissues can be fixed with a mixture of formaldehyde andglutaraldehyde or with the latter only. Glutaraldehyde fixation better preserves cellulardetails but strongly masks antigens. However, antigenic sites can be unmasked on epoxythin sections of glutaraldehyde-fixed tissues by exposing the sections to strong oxidizingagents such as EDTA, hydrogen peroxide, sodium methoxide, or sodium metaperiodate.These treatments also allow immunostaining of sections of postosmicated tissues byremoving osmium bonds. Moreover, such treatments temporarily minimize the hydropho-bicity of epoxy section surface and may increase resistance to heavy metal poststaining(Bendayan and Zollinger, 1983; Causton, 1985; Newman and Hobot, 1993).

The above-mentioned etching pretreatments are generally useful for epoxy sectionsbut not for acrylic (LR White) sections because unlike acrylic resins, epoxy resins formcovalent bonds with proteins. In other words, epoxy resins copolymerize with the tissue,while acrylic resins surround the tissue components without becoming part of them.Accordingly, epoxy resins strongly mask the proteins that become mostly inaccessible toantibodies. Therefore, epoxy sections, especially of glutaraldehyde-fixed tissues, requireetching to unmask the antigens.

The surface of acrylic sections is rougher than that of epoxy sections. Moreover,acrylic sections are less crosslinked and more hydrophilic than epoxy sections. As a result,immunostaining reagents penetrate acrylic sections easily, facilitating antigen detection.Exposure of acrylic sections to oxidizing agents worsen both the known instability of thesesections under the electron beam and the structural details.

To facilitate the access of antigens to antibodies, the protocol of embedding and etch-ing given on page 159 is used (Crowley, 1997). The sections of this low-crosslinkedembedding medium are thought to allow easy penetration of aqueous immunostainingfluids.

A saturated solution of sodium periodate is prepared by dissolving 1 g of this reagentin 5 ml of distilled water and passing the solution through a pore filter. The gridsare wetted by floating them on drops of distilled water and then floated on drops ofthe sodium periodate solution for ~15 min (this duration can be changed to obtain maxi-mum immunoreactivity). The grids are thoroughly rinsed in distilled water and must notbe allowed to dry before immunostaining.

Antigen Retrieval on Resin Sections 159

Embedding Media:Araldite 502 15mlEponate 12 25mlDDSA 55mlDibutyl Phthalate 1 %DMP-30 1.5%

If background staining is a problem and standard rinsing with PBS fails to reducenonspecific staining, boosting the sodium chloride concentration from ~0.9% (150 mM)to ~4.5% (750 mM) may help (Chiovetti, 1998). After this treatment, the grids must berinsed several times in standard PBS before further processing, so that the salt concentra-tion is reduced to the range of physiological strength. As an example, the grid can be rinsedfive times for ~2 min each on drops of high-salt buffer, followed by two rinses for ~2 mineach on drops of standard salt buffer. This routine can be used after incubation in theprimary antibody or any other incubation (e.g., secondary antibody incubation, colloidalgold) that is suspected of contributing to nonspecific background staining.

Although the exact explanation for the beneficial effect of the high-salt concentrationis not known, it may alter the conformation of protein molecules and change their overallcharge, making them less likely to bind nonspecifically on the surface of the section. It isknown that high salt concentrations tend to precipitate proteins out of the solution in bio-chemical studies and are also used to wash chromatography columns. Accordingly, onlythe antibody molecules that have been bound specifically to antigenic sites remain on thesection surface in the presence of high salt concentrations.

If cross reactivity is a problem during conjugated gold-antibody double labeling withmonoclonal antibodies from the same animal (e.g., mouse monoclonals), it can be avoidedby incubating very carefully first one side of the grid in one of the mouse monoclonals andthen the other side of the grid in the second mouse monoclonal (Chiovetti, 1998).Precaution must be used to prevent sinking of the grid in drops of the incubation reagents.Hexagonal mesh, uncoated nickel grids should be used.

To avoid the adverse effect of high temperatures on thin resin sections in themicrowave oven, staining can be carried out at ~5°C in the microwave oven (Hernández-Chavarría and Vargas-Montero, 2001). Heat generated by microwave irradiation is dissi-pated by this approach. Rapid staining is accomplished by molecular vibrations in themicrowave oven, which induce molecular collisions leading to accelerated chemical reac-tions. Thin resin sections of the tissue fixed with glutaraldehyde/osmium tetroxide aretransferred onto a grid, which is then placed into a BEEM capsule. Six capsules are placedon a plastic support, which is placed into a 500-ml beaker containing ice cubes and 300 mlof tap water, covering the bottom of the capsules. It takes ~5 min to equilibrate thetemperature in the ice bath to 5°C, which is maintained during staining in the microwaveoven. The temperature is measured after each heating period, and ice cubes are added asmelting occurs.

The staining is carried out with of 4% uranyl acetate in 50% ethanol for 1 minin a microwave oven set at a power level of 125.6W, followed by rinsing with 500 ml ofdistilled water. This is followed by staining for 1 min with of triple lead citrate (Satoet al., 1988) and then rinsing with 500 ml of distilled water. This lead citrate staining solu-tion avoids the production of artifactual lead carbonate precipitates.

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ANTIGEN RETRIEVAL ON SECTIONS OF MODIFIED EPOXY RESIN

Because epoxy resins copolymerize with tissue proteins, and acrylic resins do not, sec-tions of the former yield less immunostaining. However, to take advantage of the othersuperior characteristics of epoxy resins explained earlier, the immunostaining of sections ofthese resins can be enhanced by moderately increasing the proportion of the acceleratorDMP-30 and microwave heating (Brorson, 1998a, b; Brorson et al., 1999). Conventionalconcentrations of accelerator in the epoxy mixture form abundant chemical bonds betweenresin and tissue. In contrast, a high concentration of accelerator reduces copolymerizationof the epoxy resin with tissue proteins, while heating breaks down both protein crosslink-ages introduced by aldehydes and the bonds between the resin and the tissue. The break-down of abundant bonding with heating in the former case is insufficient to allow efficientaccess of the antibody to the antigen. Figure 7.3 shows the possible mechanism responsiblefor exposing epitopes to antibodies on the surface of thin epoxy sections after heating.

Tissue specimens are fixed overnight at 4°C with a mixture of 4% paraformaldehydeand 1% glutaraldehyde in 0.1 M phosphate buffer (pH 7.3). They are dehydrated in ethanolfollowed by propylene oxide. Infiltration is carried out in two steps using DMP-30 inconcentrations of 4% and 2%, respectively, and embedding in the resin containing 2%DMP-30. The specimens in gelatin capsules are polymerized for 3 days at 56°C. Thin sec-tions mounted on nickel grids are treated in 0.01 M citrate buffer (pH 6.0) for 15 min at95°C in a PCR machine (GeneAmp 2400, Perkin Elmer).

The sections are treated with 10% BSA in PBS (pH 7.2) for 4 hr to block nonspecificlabeling. Incubation is carried out overnight at 4°C in the primary antibody, appropriatelydiluted in PBS. This is followed by washing three times for 5 min each in PBS and incu-bation for at 22°C in colloidal gold (15 nm)–conjugated secondary antibody, appro-priately diluted in PBS containing 3% BSA. The sections are poststained with 5% uranyl

Antigen Retrieval on Resin Sections 161

acetate in 30% ethanol for 20 min and then with lead citrate for l0 min. The results of thisprocedure are shown in Figure 7.4.

EFFECT OF HEATING

Heating is effective in antigen retrieval on semithin and thin sections of resin-embeddedtissues. This results not only from the breakdown of protein crosslinks introduced by alde-hyde but also from the breakage of bonds between the epoxy resin and the embeddedtissue (see Fig. 7.2). It is known that epoxy resins form covalent bonds with tissue proteinsduring embedding.

Microwave heating has been employed for antigen retrieval on thin sections offormaldehyde and tissues embedded in Araldite for electron microscopy(Stirling and Graff, 1995). In this study thin sections on grids were treated for 1 hr at roomtemperature in a humid chamber with a saturated aqueous solution of sodium metaperiodateto reverse the effects of The heat treatment was carried out on a hot plate. Treatmentof thin sections with sodium ethoxide is not recommended, for it damages the ultrastructure.

Microwave heating has also been used for antigen retrieval on thin sections of tissuesfixed with glutaraldehyde and and embedded in LR White or TAAB resin (Wilsonet al., 1996). In this study, compared with nonmicrowaved sections, microwave-treatedthin sections revealed markedly enhanced gold labeling of type IV collagen in the oralepithelial basal lamina for both types of resins.

ANTIGEN RETRIEVAL ON THIN RESIN SECTIONSUSING AUTOCLAVING

In addition to antigen retrieval on sections of paraffin-embedded tissues for lightmicroscopy, antigen retrieval can be carried out on thin resin sections for electron

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microscopy. Using heat pretreatment, antigen retrieval can be accomplished on paraffinsections and thin resin sections. The following method was used for immunostaining thinsections of tissue embedded in a resin (Xiao et al., 1996).

Tissues are fixed with a mixture of 3% paraformaldehyde and 0.1% glutaraldehyde in0.1 M cacodylate buffer (pH 7.2) for 6 hr at 20°C. They are embedded in Lowicryl K4M at–40°C. Thin sections are mounted on uncoated metal grids or synthetic grids and air-dried. They are placed in 10 mM sodium citrate buffer (pH 6.0) and heated in an autoclavefor l0 min at 120°C. After being cooked for 20–30 min at room temperature, the sectionsare rinsed in PBS (pH 7.4). The sections are immersed in PBS containing 0.1% BSA and0.1% gelatin (PBSG) for 5 min, and then treated with 10% normal goat serum in PBSG for10 min. This is followed by incubation in the primary antibody (appropriately diluted) ina humid chamber overnight at 4°C.

The sections are rinsed in PBSG containing 0.1% Tween 20 and incubated for 1 hr incolloidal gold (15 nm)–labeled goat antimouse IgG diluted in 1:20 with PBSG containing

Antigen Retrieval on Resin Sections 163

0.1% Tween 20. Following rinsing in PBSG containing Tween 20, the sections are rinsedin PBSG and then in PBS. The sections are postfixed for l0 min with 2% glutaraldehydein PBS and rinsed several times in PBS and distilled water; poststaining is carried out withuranyl acetate and lead citrate. For negative controls, the primary antibody is replaced withPBS. The results of this procedure are shown in Figure 7.5.

RAPID STAINING OF THIN RESIN SECTIONS IN MICROWAVE OVEN

A microwave oven operating at 2,450 MHz with a maximum output power of 900 Wand a cycle time of 2 sec can be used for rapid staining in a microwave oven (Cavusogluet al., 1998). The oven contains a gas exhaust system and a built-in ceramic thermocoupletemperature probe (PT 100). To determine the distribution of microwave heating, a piece ofthermal paper is placed on the floor of the oven and subjected to microwave heating at900 W for 1 min, and the hot spots are located. A glass bottle containing 40 ml of tap wateris placed in the oven to measure the temperature during heating. The temperature of thewater is monitored throughout the staining.

Tissues are fixed with glutaraldehyde followed by and embedded in Epon. Thinsections are mounted onto a Formvar-coated grid, which is placed (section side down) onthe surface of 4% aqueous solution of uranyl acetate in a staining dish. The dish is placedon the hot spot in the oven at a power of 600 W for 1 min at 20°C (initial temperature) to94°C (final temperature). The dish is taken out of the oven and the grid is rinsed with dis-tilled water. The grid is placed on the surface of lead citrate solution in the dish, which isplaced in the oven and stained for 1 min at 20°C (initial temperature) to 93°C (final tem-perature). The results of this procedure are shown in Figure 7.6 (Cavusoglu et al., 1998).Figure 7.7 shows ultrarapid staining of biopsy heart tissue with uranyl acetate and leadcitrate for 15 sec each in a microwave oven.

MICROWAVE HEAT-ASSISTED RAPID PROCESSING OF TISSUESFOR ELECTRON MICROSCOPY

In certain diagnostic studies with the electron microscope, it is helpful to completefixation and embedding as quickly as possible. This accelerated processing can be com-pleted in ~2 hr. As shown in Figure 1.1B, the quality of cell preservation is satisfactory.The recommended protocols for routine processing, routine microwave processing, andvacuum microwave processing, respectively, are given in Table 7.1 (Giberson et al., 1997).

MICROWAVE HEAT–ASSISTED IMMUNOLABELINGOF RESIN-EMBEDDED SECTIONS

Conventional, high-resolution immunoelectron microscopy has been extensively usedfor the subcellular distribution of proteins to obtain information on their functions.However, this approach is time consuming. Processing time can be substantially reducedby applying microwave heating; the total time is reduced to 4–5 hr while the conventional

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Antigen Retrieval on Resin Sections 165

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method requires ~20 hr. All steps are carried out in a microwave oven. Tissues are fixedin a mixture of formaldehyde and glutaraldehyde, dehydrated in ethanol, and embedded inLR White resin for 75 min. Thin sections are incubated in primary antibody at 37°C for15 min and then in colloidal gold–goat antirabbit IgG for 15 min at the same temperature(Rangell and Keller, 2000).

Temperature should be strictly controlled in the microwave oven with a temperatureprobe that has a feedback mechanism to regulate the energy output of the microwave ovenand thus maintains the optimal temperature. Alternatively, temperature can be controlledby placing a water load in the chamber of the microwave oven, which absorbs extra energyand provides humidity, slowing the evaporation of reagents. In addition, hot spots in thechamber should be avoided by using the neon bulb display method (Chapter 5).

Labeling in the microwave oven is usually carried out at 37°C for 15 min. Longerdurations and higher temperatures may result in undesirable changes in antibody concen-tration and molarity of the salts and pH. After heat treatment, the sections should be keptat room temperature for at least 2 min to stabilize the antibody-antigen complexes. Thestep-by-step procedure for microwave heat-assisted immunolabeling of resin-embeddedthin sections for electron microscopy follows (Rangell and Keller, 2000):

1.

2.3.4.

5.6.7.

8.9.

10.

11.

12.

13.14.

15.16.17.18.19.

Fix the tissue in a mixture of 2% formaldehyde and 0.3% glutaraldehyde in 0.1M phosphate buffer (pH 7.2) for 40 sec at 37°C in a microwave oven.Rinse twice for 2 min each in the buffer at room temperature.Dehydrate twice for 45 sec each at 45°C in a microwave oven.Infiltrate for 15 min at 50°C with 1:1 mixture of 100% ethanol and LR white resinin a microwave oven.Infiltrate three times for 10 min each at 50°C in a microwave oven.Polymerize for 15 min at 95°C in a microwave oven using the temperature probe.Polymerize for 1 hr at 95°C in a microwave oven without using the temperatureprobe.Cut thin sections (may require 30 min).Transfer sections onto grids and float on drops of PBS containing 0.1%Tween-20 (PBST) for 5 min at 37°C in a microwave oven.Float the grids on drops of PBST containing 1 % bovine serum albumin and 0.1%coldwater fish skin gelatin (PBST+BG) for 5 min at 37°C in a microwave oven.Incubate by floating sections three times for 5 min each at 37°C on drops of theprimary antibody in PBST+BG in a microwave oven.Keep the sections for 2 min at 37°C in a microwave oven to stabilize antibody-antigen complexes.Rinse twice for 5 min each in PBST+BG at room temperature.Incubate three times for 5 min each at 37°C on drops of the secondary antibody-colloidal gold complex in PBST+BG in a microwave oven.Keep the sections for 2 min at 37°C in a microwave oven.Rinse twice for 5 min each in PBST at room temperature.Rinse twice for 5 min each in double-distilled water at room temperature.Stain in 1% uranyl acetate for 30 sec at 37°C in a microwave oven.Rinse twice for 5 min each in double-distilled water at room temperature. Thetotal duration is ~4.3 hr.

Antigen Retrieval on Resin Sections 167

MICROWAVE HEAT-ASSISTED IMMUNOGOLD METHODS

Both immunogold alone and silver-enhanced immunogold methods can be employedin combination with microwave heating for labeling antigens. The use of colloidal goldparticles as markers for antigenicity has become truly universal (Hayat, 1989–1991,2000a). The advantage of the immunogold–silver staining (IGSS) method over theimmunogold technique is that the former allows the use of very small colloidal gold par-ticles for detecting antigenicity with both light and electron microscopy (transmission andscanning electron microscopy) (Hayat, 1995). These methods are significantly more sen-sitive than standard immunoperoxidase procedures. The major benefit of combiningimmunogold methods with microwave heating is shortening the duration of incubation inthe primary and secondary antibodies. The use of microwave heating also offers the poten-tial for increasing the positive reaction product and decreasing the nonspecific backgroundlabel.

The immunogold method can also be employed in conjunction with EDTA and con-ventional heat for electron microscopy (Röcken and Roessner, 1999). In this study humanautopsy tissue specimens were fixed with a mixture of 2% formaldehyde and 2.5%glutaraldehyde and embedded in Epon. Various etching and antigen retrieval techniqueswere tested. The ideal pretreatment for achieving increased immunogold staining of amy-loid consisted of conventional heating of thin resin sections at 91 °C for 30 min in 1 mMEDTA (pH 8.0).

Immunogold-Silver Staining

Jackson et al. (1988) were the first to employ the immunogold–silver staining (IGSS)method in combination with microwave heating. They completed within minutes theincubations in primary and secondary antibodies for detecting immunoglobulins in paraf-fin sections of human tonsil. van de Kant et al. (1990) applied the same method, exceptthat resin instead of paraffin sections were used to detect bromodeoxyuridine incorporatedin cells of the mouse testis. Tissue morphology is preserved better in a resin than in paraffin.The former also allows the use of thinner sections.

Boon et al. (1989) used a similar procedure for staining beta-human chorionicgonadotropin in paraffin sections of the syncytiotrophoblast of first-trimester placenta.Recently, using the IGSS method, Taban and Cathieni (1995) visualized the gold-protein-ligand complex on cryostat sections of rat brain; this method can be used for lightand electron microscopy.

Droplet Procedure

Tissues are fixed with buffered formalin or Kryofix and embedded in paraffin (Boonet al., 1991). Sections ( thick) are transferred to a glass slide, deparaffinized, rehy-drated, and washed in running tap water for l0 min. They are treated with Lugol’s iodinefor 5 min and rinsed briefly in tap water. Following destaining with 2% aqueous sodiumthiosulfate for 10–15 sec, the sections are washed in running tap water for l0 min.

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The sections are washed in two changes of 5 min each in Tris buffer I (2.5% NaCl and0.55% Tween 20, diluted in 0.05 mol/liter Tris-HCl buffer, pH 8.2). The excess buffer isremoved by wiping around the sections, which are then covered for l0 min with ofnormal goat serum (NGS) diluted 1:1 with PBS (pH 7.4). Excess NGF is removed bywiping around the sections.

The sections are covered with of primary antibody appropriately diluted in PBS(pH 7.4) containing 0.05% BSA (freshly prepared). The slide is placed on the polystyreneplatform in the microwave oven and heated at 50% power for 5 min. A water load of 200 mltap water has already been placed in the oven. The sections are washed with Tris buffer forl0 min, followed by washing in two changes of l0 min each in Tris buffer II (0.05 mol/literTris-HCl buffer, pH 8.2). Excess buffer is removed by wiping around the sections, whichare then covered with of NGS for 10 min at room temperature.

Excess NGS is removed, and the sections are covered with of colloidal goldconjugated secondary antibody. The slide is placed on the polystyrene platform in themicrowave oven and heated at 50% for 5 min; the oven contains a water load of 200 ml oftap water. The sections are washed in three changes of 5 min each in Tris buffer II, rinsedwith distilled water, and washed three times of 3 min each in distilled water.

After excess water is removed, the sections are covered with of silverenhancement mixture for 8–11 min at 20°C. The silver enhancement mixture is preparedimmediately before use by mixing equal volumes of the enhancer and initiator solutions ofthe Janssen Intense™ LM kit. The sections are washed three times for 5 min in distilledwater, dehydrated, and mounted.

Chapter 8

General Methods of AntigenRetrieval

GENERAL PROCEDURE FOR ANTIGEN RETRIEVAL USINGMICROWAVE HEATING

(See Fig. 8.1.)1.

2.

3.4.5.

6.7.

8.

Fix the tissue without delay for 4–6 hr in 4% buffered formalin; the longer the tis-sue remains in the fixative, the lesser the chances of epitope retrieval (Hayat,2000a,b).Wash in several changes of PBS; if available, an Autotecnicon should be used inthis step and in steps 3 and 4 below.Dehydrate in a series of ascending concentrations of ethanol.Infiltrate and embed in paraffin.Cut sections with a microtome and float them on a water bath kept atroom temperature so as to stretch the sections. An ordinary glass slide is used totransfer the sections onto another water bath kept at 58°C to further stretch thesections. Lift them by the SuperFrost slides, thus mounting them in the process.The sections are allowed to dry in an upright position in a slide holder at a tem-perature of <30°C. When the slide holder is full, it is transferred to a conventionaloven.Dry the sections overnight at 58°C in the oven.Remove the sections from the oven and deparaffinize them with three changes of5 min each: in xylene, followed by three changes of 100% ethanol, two changesof 75% ethanol, and three changes of distilled water. A wash in Tris-bufferedsaline (TBS) (pH 7.3) for l0 min is optional.Place the slides in a microwave-proof (microwave-transparent) jar containing0.01 M sodium citrate buffer (pH 6.0); rectangular plastic jars are better thanglass Coplin jars. Plastic Coplin jars are commercially available (BaxterScientific, S7666). When vacuum is used in the microwave oven, use Teflon jarswith thick walls or high-quality glass jars instead of plastic jars. This jar is keptin another larger jar containing water to catch the boil-over from the smaller jarcontaining the slides. Place a jar containing the buffer or distilled water in theoven during the boiling of the slides so that when required to top up the slide jar,

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General Methods of Antigen Retrieval 171

9.

10.11.

12.

13.

14.15.16.

17.18.19.

20.

21.

22.

the liquid is at the same temperature as that of the slides. This jar also acts as neu-tral ballast in the oven, slowing down the speed at which the boiling temperatureis achieved. The buffer is prepared as follows:

Stock solution A: 0.1 M citric acid is prepared by mixing 21.01 g citric acid withenough distilled water to make 1,000 ml.

Stock solution B: 0.1M sodium citrate is prepared by mixing 29.41 g sodiumcitrate with distilled water to make 1,000 ml.The working solution is prepared just before use by mixing 18 ml of solution A

with 82 ml of solution B plus sufficient distilled water to make 1,000 ml, andadjusting the pH to 6.0.The jar containing the slides can be covered with loosely fitting lids or vented

screw caps; do not tightly close the jar or use aluminum foil to cover the jar.However, covering the jar is not obligatory if it has enough (2–3 cm) empty spaceabove the buffer level.Place the jars in the center of the oven on a rotary plate to ensure uniform heat-ing of the slides.Set the power to maximum; a power setting from 7–10 is recommended.Set the time to 10–15 min, and check the buffer temperature with a temperatureprobe. The temperature of the buffer is different from that in the oven, so it is dif-ficult to measure and control the temperature in the jar.When the buffer begins to boil, allow it to cool for 5 min. Count the time ofantigen unmasking from the boiling time. It is necessary to obtain vigorousboiling. The time for epitope unmasking depends upon the antigen, the antibody,the tissue type, the type of the fixative, and the duration of fixation. Thus, onehas to standardize microwave heating by trial and error. A known positivecontrol is essential. As an average, the time for epitope unmasking varies from2–10 min.Check the level of the buffer in the jar, and restore it between heating cycles.Compensate the buffer outflow with the buffer, while replenishing evaporatedbuffer with distilled water. The slides must remain fully immersed in the buffer.After 5 min, again set the time to 5 min and restart the oven.Repeat steps 12 and 13.Remove the jar from the oven, and allow it to cool at room temperature for20 min in a fume hood.Rinse several times in 0.05 M PBS (pH 7.5).Discard the used buffer.When the DAB method is used, inhibit endogenous peroxidase by treating thesections with of 30% in 50 ml of PBS for 30 min, followed by thor-ough washing in PBS.Treat the sections with a mixture of 3% normal serum and 0.4% Triton X-100 for~30–60 min at room temperature to aid antibody penetration and block back-ground staining.Drain the excess serum from the slides, and incubate overnight at 4°C in a humidchamber with the primary antibody diluted appropriately in PBS. Incubationshould be carried out with stirring to promote antigen-antibody contact.Rinse in three changes of PBS.

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23.

24.25.

26.27.

28.29.30.

Incubate for 30 min at room temperature in the linking agent (biotinylated anti-immunoglobulin; Vector Laboratories, Burlingame, CA).Rinse in three changes of PBS.Incubate for 45–60 min in avidin-biotin peroxidase or alkaline phosphatase orABC Elite or a third antibody if using the double indirect method.Rinse in three changes of PBS.Develop the color in 0.02–0.05% DAB activated with 0.003–0.01% in 0.1 MTris buffer for 5–15 min. This step must be carried out in a fumehood. If usingthe alkaline phosphatase method, use the Alkaline Phosphatase Developing kit(Vector Red).Wash in running tap water for 10 min.Counterstain lightly for 20 sec with hematoxylin, and rinse in water.Dehydrate in ethanol, clear in xylene, and cover-slip with Permount.

Note: Instead of coating the slides with an adhesive, use SuperFrost Plus slides (FisherScientific). These slides are charged positively and the sections are charged negatively,thus, preventing the sections from detaching from the slides while boiling. If these slidesare unavailable, ordinary glass slides can be coated with an adhesive such as poly-L-lysine(0.1%) or neoprene (Aldrich Chemical, Milwaukee, WI). However, the use of any type ofadhesive on the slide may not prevent detachment of sections from the slide. The main rea-son for losing sections during boiling is the presence of air bubbles between the sectionand the slide, not the lack of adherence of the section to the slide.

To avoid the risk of drying the sections during microwave heating, it is necessary toheat them in multiple 4- to 5-min cycles and to replenish the jars between heating periods.Some evidence indicates that drying of sections on glass slides prior to histological stain-ing in a microwave oven instead of in a conventional oven or on a hot plate, has severaladvantages: paraffin sections adhere better to the glue-coated slides, drying time is reducedfrom 1 hr to 1 min, and nonspecific background staining may be reduced.

It has been suggested that drying of paraffin sections first at 38°C and then at highertemperatures improves immunostaining of the proliferating cell nuclear antigen (Golickand Rice, 1992). Additional studies are required to evaluate the relationship between thetemperature of slide drying and the extent of immunostaining. Another suggestion is thatmild boiling of the epitope retrieval fluid gently affects tissue sections, so they are lesslikely to be dislodged from the slide. The microwave oven should be left at high rather thanchanged to a medium setting because a change of setting does not affect the wavelength oractual power of the microwaves generated.

It is thought that the intensity of specific immunostaining can be enhanced and back-ground staining simultaneously reduced by gentle orbital rotation (using a serological rota-tion) of slides during manual incubations (Butz et al., 1994). Another advantage of thisapproach is shortening antibody incubation times without sacrificing sensitivity.

Extreme antigen enhancement may cause false-positive staining. Such staining hasbeen observed in the case of p53 antigen (using monoclonal antibody D07) with TargetUnmasking Fluid (TUF) containing 35% urea in the microwave oven at 96°C for 30 min(Baas et al., 1996). This and other evidence indicates that there is a limit to the extent towhich antigen enhancement can be applied to achieve optimal detection of a given antigen.

If necessary, the slides with sections of paraffin-embedded tissues can be reused todetect a second type of antigen when the staining of the first type of antigen is negative.

General Methods of Antigen Retrieval 173

This need may arise when a limited number of slides is available. For this type of study,heat-induced antigen retrieval should be carried out only once. One such treatment lastsfor many months. Repeated use of antigen retrieval tends to cause background staining.Such an approach has been successfully used for staining epithelial membrane antigensand cytokeratin in the tonsil tissue fixed with Bouin’s fixative (Roche et al., 2000).

ANTIGEN RETRIEVAL IN ARCHIVAL TISSUES

Storage of tissues, especially nervous tissue, in the fixative impedes immunostaining.It is often difficult to obtain postmortem and other human tissues that have been in the fix-ative for a short duration. Although some antigens, such as neuropeptide Y, in brain tissueare resistant to long fixation times, the reactivity of many other antigens in this and othertissues is significantly decreased when the fixation duration is longer than a few daysbecause of excessive protein crosslinking. Examples of antigens sensitive to the effects ofa long fixation time are parvalbumin, calbindin D28-K, MAP-2, MAP-5, and the non-phosphorylated part of the neurofilament.

Physical and chemical changes such as temporary or permanent masking of epitopesoccur in tissues during routine histological processing, including formalin fixation, alco-hol dehydration, rehydration, and paraffin embedding. These changes are compounded inarchival, paraffin-embedded specimens, which cause considerable loss of immunoreactiv-ity. However, the problem is not as bleak as it may seem. Temporary loss of antigenicitydue to overfixation can be retrieved by heating. In fact, at least certain types of antigenic-ities can be retrieved with heating irrespective of the length of fixation. Some of thediagnostically and prognostically important epitopes can be detected even in specimensstored in formalin as long as 60 years (Cattoretti et al., 1992). Either microwave heatingor autoclaving is effective in epitope retrieval. Autoclaving at 120°C for 15 min, forexample, is effective for the immunostaining of steroid hormone receptors (androgen,estrogen, and progesterone) in paraffin-embedded tissue sections stored for 10 years(Ehara et al., 1996).

Another example is the study of epitopes (biomarkers) in archival paraffin blockscontaining diseased tissue such as tumors, which is important for both identifying andcharacterizing early preinvasive neoplastic lesions, for correlating their expressions withdiagnosis and prognosis of invasive tumors, and for investigating normal cellular activity(Grizzle et al., 1995; Myers et al., 1995). Identification of epitopes in archival tissues isalso helpful in identifying patients who are eligible for novel therapies, including genetherapy and immunotherapy, and monitoring the effectiveness of conventional and noveltherapies (Deshane et al., 1994).

To accomplish the aforementioned and other antigenicity detection goals, epitopesmust be detected reliably and reproducibly in archival, paraffin-embedded tissues. Sincefixation is the most important factor in preserving and masking antigenicity in archival andother specimens, a brief comment on the effects of various fixatives on epitopes is rele-vant. There is no universally ideal fixative to optimally detect all types of epitopes inarchival tissues. A few examples suffice.

Immunohistochemical studies demonstrate that unbuffered zinc formalin (a slowcrosslinking fixative) and unbuffered acid formalin yield good preservation of antigensp185 and TGF, whereas ethanol and methanol (two coagulating fixatives) produce good

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results for keratin and p53 antigens in 3-mm-thick paraffin sections of archival tissues(Arnold et al., 1996). Acid formalin is less effective as a protein crosslinking agent, as itprotonates amino groups and thus allows easy access of the antibodies to the epitopes. Inthis study, buffered formalin produced the least satisfactory results, which was expectedbecause the fixative forms protein crosslinks more rapidly and effectively. Nevertheless,note that the above-mentioned fixatives, other than buffered formalin, produce compara-tively poor morphological details.

Method for Microwave Heating of Archival Tissue Blocks

The following method is recommended for immunostaining of MAP-2, SMI-32,SMI-311, SMI-312, and the calcium-binding proteins calbindin D28-K, parvalbumin, andcalretinin in the neuroscience field (Evers and Uylings, 1994a, b, 1997). Human brains6hr after death are fixed with 4% buffered formaldehyde for 4 years. Cortical blocks(5 mm), cut from the brain, are washed for several hours in running tap water and left inTris-buffered saline (PBS) (pH 9.0). They are transferred to a plastic jar containing PBSplaced in a microwave oven, and heated at full power (boiling) for 15 min; the fluid levelis checked every 5 min. The temperature is controlled with the temperature probe. It takes~3 min to reach a temperature of 90°C.

The jar is allowed to cool for 15 min, and then the tissue blocks are placed in TBS(pH 7.6). Thick sections are cut on a vibratome, collected in plastic vials con-taining TBS, washed several times in TBS for 1 hr, and immunostained according to standardprocedures given in this volume. These thick sections allow staining of whole neurons,including neuronal processes, to distinguish different morphological types.

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ANTIGEN RETRIEVAL USING A CONVENTIONAL OVEN

Excessive treatment with microwave heating or enzymatic digestion tends to damagecell morphology and even occasionally destroy tissues, whereas insufficient treatment mayresult in false-negative results. Optimization of microwave heating is difficult. An alterna-tive simple approach is to heat the deparaffinized sections overnight in l0mM citrate buffer(pH 6.0) in a standard oven at 70–80°C (Man and Tavassoli, 1996). A variety of nuclear andcytoplasmic epitopes (e.g., estrogen and androgen receptors, p 53, Ki-67, and cytokeratin)can be demonstrated with excellent perservation of morphology, intense reactivities, andclean background. Optimal immunostaining for each of the 15 antibodies tested wasobserved even when sections from 15 different cases were heated together for a fixed dura-tion and then immunostained in an identical manner according to manufacturers’ recom-mended antibody concentrations. Thus, this method is a standard method for at least these15 antibodies. Controls included omission of the primary or secondary antibody and sub-stitution of the primary antibody with nonimmune sera or PBS. No positive immunostain-ing was observed in any of the negative controls. The only drawback of this approach is thatit takes longer to yield results. The results of this method are shown in Figure 8.2 (Plate 3G).

HOT PLATE–ASSISTED ANTIGEN RETRIEVAL

In certain cases antigen retrieval in formalin-fixed and paraffin-embedded tissues ismore efficient using conventional heating with a hot plate than that using standard microwaveheating or pepsin predigestion. In such cases, the hot plate method yields strongerimmunostaining even in tissues that have been fixed with formalin for as long as 1 month.This advantage is important when tissues have to be stored in formalin on a weekend orduring holidays or transported to a service laboratory.

The use of hot plate heating becomes essential when the diagnosis of malignancy isbased on a negative immunoreaction. This is exemplified by the malignant prostate gland.It is known that anti-high-molecular-weight cytokeratin (HMCK) monoclonal antibodyclone selectively stains the basal layer of the prostatic duct–acinar system in thebenign prostate. In contrast, malignant glands lack immunoreactivity with this antibody(Wojno and Epstein, 1995). This antibody is especially sensitive to formalin fixation anddifferent antigen retrieval methods.

A recent study demonstrates that hot plate heating is better than other antigen retrievalmethods for detecting even weak HMCK positive staining in the radical prostatectomyspecimens fixed with formalin for 6 hr to 1 month (Varma et al., 1999). Although formalinfixation for 6 hr is the optimal duration of fixation, there is no decrease in HMCKimmunoreactivity in tissue fixed for 1 month when hot plate heating is used. This advantageis shown in Figure 8.3; the cells were fixed for 54 hr. The above and other evidence rein-force the necessity for standardized fixation and antigen retrieval method in each laboratory.

Procedure

Sections thick) of formalin-fixed and paraffin-embedded tissues are mountedon gelatin-coated slides, which are placed in a beaker containing 1,000 ml of 0.2 M sodium

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citrate buffer (pH 6.0) and heated for l0 min at 100°C on a hot plate (PC-351) (Corning,Utica, NY). The slides are allowed to cool at room temperature for 20 min. After beingwashed in PBS, the sections are incubated overnight at 4°C in the monoclonal antibodyclone E12 (DAKO, Carpinteria, CA) and diluted 1:4000 with PBS. They are washedwith PBS and then treated with the Elite ABC system according to the directions of themanufacturer (Vector, Burlingame, CA). The chromogen is developed with 3-amino-9ethyl carbazole (Sigma) at room temperature. After being washed with PBS, the sectionsare counterstained with Mayer’s hematoxylin and mounted in Glycergel (Sigma). Theresults of this procedure are shown in Figure 8.3.

HOT PLATE–ASSISTED GRADING OF VULVAR INTRAEPITHELIALNEOPLASIA

The grade of vulvar intraepithelial neoplasia (VIN) can be scored by subclassifyingit on the basis of the extent of cellular changes into three types (Van Beurden et al., 1999):

1.2.3.

VIN 1 (cellular disarray involves the lower two-thirds of the epithelium)VIN 2 (cellular disarray involves more than the lower two-thirds of the epithelium)VIN 3 (cellular disarray involves more than the lower two-thirds of the array)

Vulvar intraepithelial neoplasia show a spectrum of pathological alterations, includingnuclear pleomorphism, hyperchromasia, altered epithelial maturation, cellular aggrega-tion, loss of normal keratinocyte polarity, and atypical mitotic features (Wilkinson, 1992).

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Conventional light microscopy of multifocal lesions of VIN may not show VIN 3 andinstead may show VIN 2, VIN 1, or even normal squamous epithelium (van Beurden et al.,1998). Interobserver variation in the interpretation of the grading of VIN is not uncommon.To determine correct treatment of the patient, it is necessary to know which lesions showVIN 3 and which do not. The standard treatment for VIN 3 is drastic surgical excision ofall visible lesions. However, an alternate approach is taking multiple biopsies; the removalof involved skin using cold knife surgery or laser vaporization without radical surgery (vanBeurden et al., 1998).

Interobserver variation in the grading of VIN has been observed when usinghematoxylin-eosin only, MIB-1 monoclonal antibody alone, or combined hematoxylin-eosin and MIB-1 antibody (van Beurden et al., 1999). This antibody is the most versatileproliferation worker for the sections of formalin-fixed and paraffin-embedded tissues.Normal vulvar skin and VIN lesions are fixed with formalin and embedded in paraffinaccording to standard procedures.

Following deparaffinization with xylene and rehydration with ethanol, the slides areplaced in 0.01 M sodium citrate buffer (pH 6.0) and boiled for l0 min on a hot plate; aftercooling for 20 min to room temperature the slides are subjected to three different treatments:

1.2.3.

Staining with hematoxylin-eosinIncubation in MIB-1 antibodyIncubation with a combination of MIB-1 antibody and hematoxylin-eosin

Uncertainty regarding the grading of VIN is significantly decreased when MIB-1antibody is used.

WATER BATH HEAT–ASSISTED ANTIGEN RETRIEVAL

Although the microwave heat–assisted antigen retrieval method is widely used inclinical pathology, it has several limitations. The rise of temperature in the microwave ovenis difficult to control. Unintentional, very high heating of sections in the oven is notuncommon. Thus, standardization of temperature in the oven becomes difficult. Very hightemperatures tend to damage cell morphology. Microwave heating is especially damaging tofree-floating sections and causes wrinkling, among other problems. Such sections can alsobe expelled from the antigen fluid during vigorous bubbling at high temperatures in themicrowave oven. Limitations of microwave heating have also been discussed on page 142.

One of the alternatives to the microwave heating method is the water bath heatingtechnique. The latter is simple and inexpensive and allows precise temperature control(including uniform rise in temperature), which enables the achievement of reproducibleantigen retrieval as well as other histological data. The water bath can be heated in a con-ventional oven; alternatively, heated water baths are commercially available. The subboil-ing water bath heating (80°C) method is effective in the antigen retrieval on free-floatingsections, cryostat sections, and paraffin-embedded sections of tissues fixed with formalde-hyde and/or glutaraldehyde (Jiao et al., 1999). The advantage of using glutaraldehyde is itsexcellent preservation of the ultrastructure using electron microscopy. The water bath heat-ing technique can also be used for immunofluorescence microscopy. This technique is espe-cially useful for processing free-floating sections of brain tissue. The following methods

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are recommended for immunostaining on thick and thin sections using light and electronmicroscopy, respectively, and also for cell smears.

Procedure for Electron Microscopy

The animal is fixed by vascular perfusion with a mixture of 3% paraformaldehydeand 0.1% glutaraldehyde in 0.15 M PBS (pH 7.4) according to standard procedures. Thebrain (or another organ of interest) is removed and tissue blocks containing the region ofinterest are refixed overnight with the same fixative. The blocks are agar-embedded andcut into sections with a vibratome. The sections are washed with PBS andthen with saline containing 10mM sodium citrate (SSC) (pH 8.5). They are placed in SSCand heated in a water bath for 30 min at 76–80°C. The sections are cooled in SSC to roomtemperature for l0 min and then washed with PBS. The sections are treated with 1%sodium borohydride in PBS for 15 min and then thoroughly washed with PBS. They areincubated in 4% normal serum in PBS for 1 hr, using serum other than that used to gener-ate the primary antiserum. For example, if primary antiserum from rabbit is to be used,tissue should be prerinsed in normal goat serum (NGS). In the following procedure, eachstep is followed by a rinse in PBS, unless otherwise indicated.

The sections are incubated for ~72 hr at 4°C with primary antiserum in PBS con-taining 2% normal serum and 0.25% gum arabic (Sigma). The sections are then incubatedovernight at 4°C with a biotinylated secondary antibody (Vector Laboratories, Burlingame,CA), diluted 1:200. They are next incubated overnight at 4°C with avidin-biotinylated per-oxidase complex (ABC) diluted 1:100 in PBS containing 0.25% gum arabic. Note that theperoxidase-antiperoxidase procedure can also be used.

The bound peroxidase is visualized by reaction with a filtered solution of 0.05% DABand 0.0005% hydrogen peroxide in PBS. After rinsing with 0.15 M sodium phosphatebuffer (pH 7.4), the sections are postfixed with 0.25% osmium tetroxide in the same bufferfor 1 hr and counterstained with 1% uranyl acetate in deionized water. The sections aredehydrated and flat-embedded in Epon according to standard procedures (Hayat, 2000a).Controls are processed as above, except that incubation in the absence of the primary anti-body is carried out with PBS containing 2% normal serum and 0.015% Triton X-100.

The sections are observed under a light microscope and representative DAB-labeledneurons, and neuronal processes in the region of interest are selected for study with theelectron microscope. The sections are trimmed with a razor Wade into small pieces con-taining the immunoreactive neurons. Thin sections are cut for electron microscopy. Theresults of this procedure are shown in Figure 8.4.

Procedure for Light Microscopy

Human brain tissues, for example, are fixed postmortem with 10% formalin for 24–48 hr,dehydrated, and embedded in paraffin. Sections, cut with a rotary microtome, are mountedon coated glass slides. The sections are rinsed three times for 5 min each with 0.1 Msodium phosphate buffer (pH 7.4) and then transferred to 10–15 mM sodium citrate

General Methods of Antigen Retrieval 179

(pH 8.5–9.0; preheated to 80°C in a water bath). They are cooled to room temperature, fol-lowed by rinsing three times for 5 min each in the buffer. The sections are immersed in0.3% nonfat dry milk in buffer containing 0.3% Triton X-100 and 0.01% sodium azide for30–60 min.

The sections are incubated in a primary antibody (diluted appropriately) for 72 hr at4°C in a sealed humid chamber; the incubation is carried out by applying droplets of theantibody to the sections. After being rinsed in the buffer, the sections are incubated for90 min in secondary antiserum diluted 1:50 with PBX (0.3% Triton X-100, 0.01% sodiumazide and 0.1 M phosphate buffer) and then treated for 1 hr under agitation in peroxidase-antiperoxidase (PAP), diluted 1:100 with PBX, in a sealed humid chamber in both cases.

The sections are rinsed sequentially with the buffer and then distilled water. They areincubated for 10 min with continuous agitation in 50ml of 0.05 M imidazole/0.05 Mcacodylate buffer (pH 7.2) containing 50 mg of DAB, followed by an additional 10-minincubation after adding of 3% hydrogen peroxide with continuous agitation. Afterbeing washed in distilled water, the sections are placed in the buffer, dried, dehydrated,and cover-slipped with Permount. Note that the ABC procedure can also be used forimmunolabeling.

For assessing the antigen retrieval effectiveness, the staining intensities observedunder the microscope can be divided into five grades: + + + +,+ + +,+ +,+ or – for

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very intense, intense, moderate, weak, or negative, respectively. These judgments are madequalitatively by comparing one section to another.

Procedure for Free-Floating Sections

Free-floating sections of paraformaldehyde-fixed tissues are rinsed threetimes for 5 min each in 0.1 M sodium phosphate buffer (pH 7.4) (Jiao et al., 1999). Theyare transferred to 10–15 mM sodium citrate buffer (pH 8.5–9.0) preheated in a water bathkept in a conventional oven at 80°C for 30 min. The sections are allowed to remain in thisbuffer for 30 min to cool to room temperature. Following rinsing three times for 5 min eachin the same buffer, the sections are treated by immersion in 0.3–3% nonfat dry milk in0.1 % sodium azide for 30–60 min. The sections are then incubated in the primary antibody,diluted with a mixture of 0.3% Triton X-100, 0.01% sodium azide, 0.1 M sodium phos-phate buffer (pH 7.4) (PBX), and 5% normal horse serum for 72 hr at 4°C under constantagitation.

The sections are rinsed in the same buffer and incubated for 90 min in secondary anti-serum (Jackson ImmunoResearch Laboratories, West Grove, PA), diluted 1:50 with PBX-5% normal horse serum. They are rinsed in 0.1 M sodium phosphate buffer and thenincubated for 1 hr in peroxidase antiperoxidase (PAP), diluted 1:100 with PBX. The sec-tions are rinsed three times for 5 min each in the same buffer followed by distilled waterrinses. They are incubated for 10 min in 50 ml of 0.05 M imidazole/0.05 M cacodylatebuffer (pH 7.2) containing 50 mg of DAB. This is followed by an additional 10 min incu-bation after adding All incubations are carried out under constant agitation.The sections are washed in distilled water, placed in the same buffer, mounted onto gela-tin-coated slides, dried, dehydrated, and cover-slipped with Permount. Note that the ABCprocedure can be used instead of the PAP procedure.

MICROWAVE HEAT–ASSISTED EVALUATION OF GLOBAL DNAHYPOMETHYLATION

Loss of methyl groups in DNA is not uncommon in human carcinomas such as colonadenomas and adenocarcinomas. A strong correlation is found between the malignant phe-notype and DNA methylation. It is known that 5-methylcytidine (a spontaneous frequent siteof C and T mutation) is involved in the control of gene expression in carcinogenesis and intumor progression. Consequently, global DNA hypomethylation could induce protoonco-gene expression, whereas hypermethylation could silence tumor suppressor gene (Little andWainwright, 1995). Monoclonal antibodies can be used to recognize the presence ofa methyl group on the C of cytidine to investigate DNA methylation in situ. An immuno-histochemical method has been reported for correlating the histopathological pattern withthe immunostaining intensity of the nuclei (Hernández-Blazquez et al., 2000).

Qualitative and quantitative differences can be observed and measured between thenormal and malignant part of each tumor specimen. Morphologically altered nuclei dis-play densely labeled spots within faintly labeled areas, whereas normal nuclei are darkerand uniformly stained.

General Methods of Antigen Retrieval 181

Procedure

Malignant lesions and normal tissue biopsies (colon) are fixed with formalin andembedded in paraffin (Hernández-Blazquez et al., 2000). Paraffin sections thick)are deparaffinized, rehydrated, and rinsed in PBS. They are placed in 0.1 mM citrate buffer(pH 3.4), heated for 10 min in a microwave oven at full power (720 W), and washedin PBS. The slides are immersed in 2N HC1 for 2 hr at 37°C and then rinsed in PBS.The sections are covered with of hybridoma supernatant containing anti-5-MeCydmonoclonal antibody and incubated for 1 hr at room temperature with abiotinylated goat antimouse secondary antibody diluted 1:200 in PBS containing0.1% BSA.

The sections are rinsed in PBS and then treated for 30 min with streptavidin-peroxidase,diluted 1:100 with PBS-BSA. They are rinsed in PBS and treated with for5 min. The PBS used for rinsing contains 0.1% Tween 20.

MICROWAVE HEAT–ASSISTED ENHANCED PEROXIDASEONE-STEP METHOD

The enhanced peroxidase one-step (EPOS) method is considered superior to standardABC technique in that the former is more sensitive than the latter. It is known that theKi-67 antibody can only be used on fresh or frozen tissues, whereas the monoclonal anti-body MIB-1, developed against a part of the Ki-67 antigen molecule, can be used on sec-tions of formalin-fixed and paraffin-embedded tissues using antigen retrieval. Recently,EPOS Ki-67 antibodies were developed which consist of antibody molecules and horse-radish peroxidase bound covalently to dextran (Bisgaad et al., 1993). This method has beenapplied for localizing PCNA and Ki-67 antigens (Tsutsumi et al., 1995).

More recently, the EPOS protocol was compared with the standard ABC techniquefor detecting Ki-67 antigens in pituitary tumors (Turner et al., 1999). Both methods wereapplied in conjunction with microwave heating. This study demonstrates that the EPOSmethod is both more convenient and more accurate in showing the number of cells thathave entered the cell cycle. It can be inferred that the EPOS approach, in addition, detectsvery small amounts of Ki-67 antigens present in the cells in early stage, which the ABCmethod does not detect. Thus, the EPOS antibody may identify those tumors that arepotentially aggressive and require closer monitoring.

Procedure

Surgically removed tissues are fixed with 4% buffered formalin and embedded inparaffin (Turner et al., 1999). Sections thick) on slides are deparaffinized, rehy-drated, and then treated with 3% to block endogenous peroxidase. They are placed in0.1 M sodium citrate buffer (pH 6.0) and heated in a microwave oven. An EPOS rabbit anti-human Ki-67 antibody is applied as supplied (DAKO); it is ready to be used without anydilution. Color development is accomplished with metal-enhanced DAB for 15 min, fol-lowed by light counterstaining with hematoxylin. Quantification of Ki-67 antibody-labeled

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cells is performed with an image analysis system (VIDAS 21), and the tumor cell nuclearstaining is recorded as the percentage of positive cells (labeling index).

MICROWAVE HEAT–ASSISTED IMMUNOSTAINING OF CELL SMEARS

Consistent and reliable immunostaining of cytological preparations can be obtainedby ensuring that the following conditions are met (Leong et al., 1999b): (1) availability ofsufficient specimens for examination, (2) appropriate panel of diagnostic antibodies,(3) preparation of thin and uniformly spread smears, (4) removal of proteinaceous back-ground fluid and red blood cells, (5) use of optimal fixation, and (6) application of asensitive method of immunostaining. The concentration of the reagents including antibod-ies and the duration of incubation for cytological smears are lower and shorter, respec-tively, than those used for paraffin sections. These conditions must be determined by trialand error for each type of new antibody employed.

Thin, uniformly spread cell smears are prepared on glass slides and then rehydratedwith normal saline for ~3 min. This is followed by air-drying for 24 hr and fixation with0.1% formal saline (1,000ml of normal saline and 2.5ml of 40% formalin) for 2–14 hr;postfixation is accomplished with 95–100% ethanol for 10 min. The sections are heated in10 mM citrate buffer (pH 6.0) in a microwave oven for ~5 min at boiling and then allowedto remain in the hot solution for another 5 min before being removed for immunostaining.

DOUBLE IMMUNOSTAINING USING MICROWAVE HEATING

The following method was used for double immunostaining of fast and slow skeletalmuscle fibers in the same section (Carson et al., 1998). Muscle specimens (2–4 mm) arefixed in 10% neutral buffered formalin for 3 hr and then immersed in 0.1 M PBS contain-ing 17% sucrose for 3 hr. They are embedded in paraffin, and 4 to sections aremounted onto Superfrost Plus slides. The sections are deparaffinized and placed in TargetUnmasking Fluid (TUF) preheated in a microwave oven and maintained at 90°C for15 min (without boiling) and slowly cooled to room temperature. The sections are rinsedthree times for 3 min each in PBS and then placed in 3% hydrogen peroxide in PBS for30 min to block endogenous peroxidase, followed by again rinsing three times in PBS.

The sections are blocked with 10% nonimmune goat serum for 30 min at room tem-perature and then rinsed in PBS. They are incubated overnight at 4°C in monclonal anti-body against MHC-I (diluted 1:20 in PBS) in a humid chamber, followed by rinsing threetimes in PBS. The sections are incubated for 1 hr in the goat antimouse biotinylatedsecondary antibody in a humid chamber.

The sections are treated for 1 hr with streptavidin-alkaline phosphatase in a humidchamber and then washed in PBS. They are developed for 10 min in nitroblue tetra-zolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP). After washing in PBS, thesections are treated for 30 min in a double-staining enhancer (Zymed Laboratories, SanFrancisco, CA) to prevent the first stain from reacting with the second. They are thor-oughly rinsed in distilled water followed by PBS.

The sections are treated for 30 min with 10% nonimmune goat serum and then rinsedin PBS. This is followed by overnight incubation at 4°C in monoclonal antibody against

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MHC-II (diluted 1:10 in PBS) in a humid chamber and rinsing in PBS. The sections areincubated for 1 hr in the goat antimouse biotinylated secondary antibody in a humid cham-ber. They are treated for 1 hr with streptavidin–alkaline phosphatase in a humid chamberand then washed in PBS. The sections are treated with 3-amino-9-ethyl-carbazole (AEC)chromogen; the staining intensity is monitored microscopically. The staining durationis ~10 min. They are counterstained for 10 min in NBT/BCIP, washed in distilled water;and cover-slipped with an aqueous mounting medium. An immunostaining kit (Histostain-SP) is commercially available (Zymed Laboratories, San Francisco, CA).

MICROWAVE HEAT–ENHANCED DOUBLE IMMUNOSTAINING OFNUCLEAR AND CYTOPLASMIC ANTIGENS

Simultaneous, double immunostaining of two antigens in single cells in sectionsof formalin-fixed and paraffin-embedded archival tissues can be carried out. This isaccomplished by using microwave heating to detect otherwise undetectable nuclear anti-gens, followed by the labeled avidin-biotin (LSAB) procedure and the alkaline phos-phatase (APAAP) protocol to detect cytoplasmic or membranous antigens (Bohleet al., 1997).

Procedure

Sections of formalin-fixed and paraffin-embedded tissues on glass slides are deparaf-fmized in xylene (10 min), acetone (10 min), acetone/TBS (1:1, 10 min), and TBS (pH 7.4,10 min). The tissues are fixed with 4% formalin and can be stored for up to 10 years.Endogenous peroxidase is blocked in methanol containing 0.5% for 10 min.Microwave heating is carried out by placing the slides in microwave-proof tubes contain-ing 0.1 M sodium citrate buffer (pH 6.0) and boiling for 5 min at the 800 W setting. Thetubes are refilled, and the heating is repeated four times. The slides are allowed to cool toroom temperature and washed in TBS.

Incubation is carried out with primary antibody in a humid chamber for 30 minat room temperature. After being rinsed three times in TBS, the slides are incubated withrabbit antimouse antibody (1:200) in TBS for 30 min at the same temperature. They arerinsed three times in TBS and incubated with streptavidin/HRP P397 (DAKO) for 30 minat room temperature. Following rinsing three times in TBS, the slides are incubated for4 min in DAB chromogen solution.

Thereafter, of the second antibody is placed on the slide and incubated in ahumid chamber for 30 min at room temperature. After being rinsed three times in TBS, theslides are incubated with rabbit antimouse (“link”) antibody (1:25) in RPMI 1640 Medium(Life Technologies, Faisley, Scotland) for 30 min at room temperature. They are rinsedthree times in TBS and incubated with the APAAP-complex (DAKO, 1:25 in RPMI1640 Medium) for 30 min at the same temperature. Following rinsing three times inTBS, the incubation with the APAAP-complex is repeated three times for 10 min each.After being rinsed 10 times in TBS, slide development is carried out in new fuchsinchromogen solution for 30 min. The results of this procedure are shown in Figure 8.5(Plate 3H).

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MICROWAVE HEAT–ASSISTED IMMUNOHISTOCHEMICALLOCALIZATION OF CYCLIN D1

Cyclin D1 (PRAD-1, bcl-1) protein plays an important role in the transition of cellsfrom resting phase to DNA replication phase. This protein has oncogenic properties, andits overexpression is thought to play a role in tumorigenesis. Overexpression of cyclin D1mRNA and protein has been demonstrated in a variety of lymphoid as well as nonlym-phoid neoplasms (de Boer et al., 1995; Vasef et al., 1997a, b; Naitoh et al., 1995). Theoverexpression of cyclin D1 protein has also been demonstrated in neoplastic proliferatingparathyroid tissue, adenomas, and nonneoplastic proliferating parathyroid gland, but sel-dom in normal parathyroid tissue (Vasef et al., 1999). It is suggested that PRAD-1 genealteration is responsible for cyclin D1 protein overexpression in parathyroid hyperplasia.The mechanism underlying such gene alterations remains undefined. Although cyclinD1 is not useful in distinguishing parathyroid carcinomas from parathyroid adenomas,this protein is useful in distinguishing between hyperplasia and normal parathyroidglands in histologically ambiguous cases. The following immunohistochemical method isrecommended for the localization of cyclin Dl in neoplastic parathyroid tissue (Vasefet al., 1999).

Biopsy tissue specimens are fixed with formalin and embedded in paraffin. Sectionsthick) are mounted on silanated slides, heated at 56°C for 1 hr, deparaffinized in

xylene, rehydrated in graded ethanols, and rinsed in distilled water. They are placed in 0.01M citrate buffer (pH 6.0) and heated in a microwave oven for six cycles of 5 min each, fol-lowed by cooling at room temperature for 20 min. Endogenous peroxidase activity isblocked with hydrogen peroxide in distilled water for 8 min, and nonspecific backgroundstaining is prevented by treatment with nonimmune horse serum for 20 min.

General Methods of Antigen Retrieval 185

The sections are incubated overnight at 4°C in anticyclin D1 monclonal antibody(P2D11F11) (Novocastra/Vector, Burlingame, CA), diluted 1:10; this antibody does notshow cross-reactivity with cyclin D2 protein. After rinsing with PBS, the reactivity isdetected using mouse immunoglobulin G as the secondary antibody with an avidin-biotinprocedure and DAB as the chromogen. Sections of normal tonsil and known casesof cyclin D1–positive mantle cell lymphoma are used as negative and positive externalcontrols, respectively. Cases are interpreted as cyclin D1 positive if more than 10% of cellsshow positive nuclear staining, while cases with patchy clusters of positive cellsare defined as focally positive. The results of this procedure are shown in Figure 8.6(Plate 4A).

MICROWAVE HEAT–ASSISTED IMMUNOFLUORESCENCESTAINING OF TISSUE SECTIONS

Frozen sections, as well as sections of formaldehyde-fixed and paraffin-embeddedtissues, can be used for direct or indirect immunofluorescence staining after antigenretrieval using microwave heating. Although frozen sections yield higher sensitivity thanthat obtained with paraffin sections, the latter approach is necessary for retrospective stud-ies of archival specimens. Moreover, fresh tissue is not always available.

Antigen retrieval using trypsin digestion in conjunction with indirect immunofluo-rescent staining in various tissues was first reported by Huang et al. (1976). The 2-hrdigestion in this study was too severe a treatment and adversely affected cell morphology.An improved antigen retrieval procedure consists of microwave heating in urea (6 M),followed by indirect immunofluorescence staining (D’Ambra-Cabry et al., 1995).This method produces more intense immunofluorescence staining than does trypsiniza-tion. Antigen retrieval can also be obtained by combining trypsin digestion with micro-wave heating, followed by direct immunofluorescent staining (Al-Rifai et al., 1997). Thisprocedure is given below.

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Procedure

Sections thick) of formalin-fixed and paraffin-embedded tissues are depositedon a coated slide, deparaffinized, rehydrated, and rinsed in PBS. They are placed in sodiumcitrate buffer-containing plastic jars and heated twice for 5 min each in a microwave oven.Following cooling at room temperature for 20 min, the sections are treated with 0.3%trypsin. They are washed in PBS, blocked with normal serum for 10 min, and then over-laid with fluorescein-conjugated rabbit antibodies to human IgG at 1:20 dilution in PBSfor ~45 min at room temperature.

The sections are washed in PBS for 5 min and incubated for ~30 min in fluoresceinisothiocyanate (FICT)-labeled swine antirabbit immunoglobulin conjugate at 1:20 dilutionin PBS. After being washed in PBS for 5 min, the sections are mounted with an aqueousmounting medium. The sections are observed under an epiilluminating fluorescent micro-scope. The negative control is not incubated with the primary antibody, and a frozen sec-tion from a patient with the known condition is used as the positive control.

MICROWAVE HEAT–ASSISTED DOUBLE IMMUNOFLUORESCENCELABELING

Most applications of immunohistological labeling to routine tissue sections focus ona single antigen. However, some studies require simultaneous labeling of two antigens.Double labeling, for example, is required to determine if two antigenic markers areexpressed in the same cell and/or two antigens are present at the same site such as twomarkers at the cell surface. The enzyme-based methods (peroxidase and alkaline phos-phatase) are rarely suitable for detecting two antigens present at the same site because onelabel tends to obscure the other. The reaction product of the antigen of a higher densitymay mask the reaction product of the antigen of a lower density. Consequently, these meth-ods are largely restricted to the detection of pairs of antigens found either at different siteswithin a single cell (e.g., nucleus and cell surface) or in different cell populations.

Double immunofluorescence labeling in conjunction with microwave heating can beused to visualize two markers at the same cellular location in routine formalin-fixedand paraffin-embedded tissue sections (Mason et al., 2000). The primary antibodies areeither monoclonal antibodies of differing isotype/subclass or antibodies from differentspecies. Labeling is visualized on a conventional fluorescence microscope equipped witha cooled analog monochrome CCD camera (Model C 5985, Hamamatsu Photonics,Billerica, MA) and recorded using “off the shelf” personal computer hardware and soft-ware. Contrary to general belief, paraffin-embedded tissue sections do not show excessivenonspecific fluorescence.

Procedure

Tissues are fixed in formalin, embedded in paraffin, and sections thick) are trans-ferred onto Superfrost Plus–coated slides (Mason et al., 2000). The sections are deparaf-finized with xylene and then rehydrated with descending concentrations of ethanol. Theyare placed in 0.1 M sodium citrate buffer (pH 6.0) and heated in a microwave oven at full

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power. The sections are rinsed in PBS and then incubated with a mixture of two primaryantibodies for ~30min at room temperature, using appropriate dilutions that have beendetermined by titration. The pair of primary antibodies are either from different speciesor of differing Ig isotypes/subclasses. They are washed in TBS and incubated for 1 hr inthe dark in secondary antibodies, one conjugated to fluorescein isothiocyanate (FITC) andthe other to Texas Red. This is followed by washing in TBS and then counterstaining for30 sec with DAPI (1 mg/ml of an aqueous solution diluted 1:100 in absolute ethanol). Thesections are rinsed in tap water and mounted in antifading medium (DAKO). The slidesshould be kept at 4°C in the dark when not viewed immediately.

MICROWAVE HEAT–ASSISTED DOUBLE INDIRECTIMMUNOFLUORESCENCE STAINING

Microwave heating is useful for double indirect immunofluorescence staining, pro-vided excessive heating is avoided. Moderate microwaving neither elutes antibodies norleads to detectable loss of fluorescence and prevents their reactions with subsequentlyapplied reagents. Prolonged staining will elute the antibodies. Moderate heating applied inbetween the first and second staining cycles facilitates double indirect immunofluores-cence staining of antigens using primary monoclonal antibodies raised in the same species.In addition, heating inhibits reactions with endogenous immunoglobulins present in extra-cellular compartments, which substantially reduces background staining but does notblock the staining of the plasma cells and/or lymphocytes.

The above procedure has been applied for double indirect staining of mouse pancre-atic tissue using fluorescein isothiocyanate (FITC)–conjugated antimouse IgG and TexasRed (Tornehave et al., 2000). Although this method has been successfully used by theseauthors, it may require amendment for other studies. Before commencing the double stain-ing studies, the potential unmasking effect of microwaving on individual antigen-antibodycombinations should be tested because repeated cycles of microwaving will progressivelyelute the fluorescence-labeled antibodies. With immunoenzymatic detection, this concernis irrelevant as long as the enzyme reaction products remain after microwaving.

Procedure

1.

2.

3.

The animal is perfused first with 1–2 ml of saline, followed by 4% paraformalde-hyde in 0.1 M sodium phosphate buffer (pH 7.4). Postfixation is accomplishedovernight at 4°C in the same fixative. Alternatively, the tissue can be fixed byimmersion in the same fixative or 10% commercial formalin in the same buffer.For cryostat sectioning, the tissue specimens are cryoprotected in 30% sucrose in0.1 M phosphate buffer for 12 hr or until they sink to the bottom of the container.They are embedded in O.C.T compound (Miles, Elkhart, IN) and frozen in N-heptane cooled to the temperature of liquid nitrogen. Alternatively, if the antigensare resistant to paraffin embedding, the specimens can be dehydrated in gradedethanol, cleared in xylene, and embedded in paraffin.Sections ( thick) are hydrated and treated with 1 % bovine serum albumin orwith 10% serum from the same species from which the second antibody is derived

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(diluted in 0.05 M Tris buffer [pH 7.4] containing 0.15M NaCl: TBS) for 30 minat room temperature.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

Antisera or monoclonal antibodies are applied by diluting as required in TBScontaining 0.25% BSA (TBS-BSA). Monoclonal antibodies are applied for 1 hrat room temperature, while polyclonal antibodies are applied overnight at 4°C.The sections are rinsed three times for 5 min each in TBS containing 1% TritonX-100. The addition of this detergent is optional but may sometimes reduce back-ground staining.Fluorescence-labeled second antibody is applied for 30 min at room temperatureby diluting in TBS-BSA as required. Fluorscein isothiocyanate-(FITC)–TexasRed– or aminomethyl coumarin (AMCA)–labeled variants can be used.The sections are rinsed three times for 5 min each in TBS with or without 1%Triton X-100. If this detergent is used, the sections are rinsed three times for 5min each in TBS without the detergent before microwaving.Place five slides with sections in a plastic jar containing 50 ml of 10 mM sodiumcitrate buffer (pH 6.0). The jar is placed in a tray containing 1 liter tap water andmicrowaved at 780 W. Three cycles of microwaving for 5 min each are adequate.More cycles may lead to losses of antibodies from the sections and fewer cyclesmay cause inefficient blocking of antibody cross-reactivity. The optimal condi-tions of microwaving differ depending on the antibody-antigen under study.After microwaving, the slides are left in the citrate buffer at room temperature for20 min.The sections are rinsed in TBS and then incubated in the primary antibody, whichmay be raised in the same species as those used in step 1 above.This is followed by rinsing three times for 5 min each in TBS with or without 1%Triton X-100 (cf. step 5).A new round of second antibodies labeled with a fluorophore other than that instep 6 is applied. Alternatively, a triple-layer method employing a second layerof biotin-labeled antiimmunoglobulins followed by fluorescence-labeled strepta-vidin can be used.They are rinsed three times for 5 min each in TBS with or without 1% TritonX-100. If the detergent is used, at least the final rinse should be in TBS withoutthe detergent.The sections are mounted in a suitable antifade medium such as VectaShield(Vector Laboratories, Burlingame, CA).They are observed with a fluorescence microscope equipped with selective filtersfor the fluorophores used. An example of the double staining is shown in Figure 8.7(Plate 4B, C, D).

Control Procedures

In addition to conventional staining and absorption controls, the following procedureis recommended. Parallel sections are processed through the above protocol up to step 10.Instead of specific antibody/antiserum, normal serum from the same species is applied.Steps 11–15 are carried out as described above. These control sections must not show the

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fluorescence characteristic of the label introduced in step 12. If they do, microwaving isinsufficient and has not completely blocked the free antigen combining sites presented onthe second antibodies introduced in step 6. These sites, if not blocked by microwaving, willbind to immunoglobulins present in the normal serum. These immunoglobulins in turn willbind the second round of second antibodies. Thus, all sites marked by the first antibodywill fluoresce with a mixed color characteristic of the two fluorophores used in steps 6 and12, respectively. Alternatively, it is possible that the first round of second antibodies maynot have saturated all binding sites on the primary antibodies, leaving these free to bindsecond antibodies added in the second staining cycle. In any case, successful microwavingblocks cross-reactivity.

Immunoenzymatic Detection

This procedure is also applicable to immunoenzymatic staining as originallydescribed by Lan et al. (1995). However, with immunoenzymatic detection it is oftendifficult to discern if different antigens reside in the same cellular compartment. On theother hand, with immunofluorescence, problems with interpreting mixed colors are notencountered because selective filters are used for the individual fluorophores (Larsson,1988; Tornehave et al., 2000).

COMBINED MICROWAVE HEATING AND ULTRASOUNDANTIGEN RETRIEVAL METHOD

The combined heat-induced epitope retrieval (HIER) and sonication-induced epitoperetrieval (SIER) methods have been employed for retrieving cyclin D1/bc l-1 epitope in

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mantle cell lymphoma (Brynes et al., 1997). Compared with HIER or SIER method alone,the combined approach produced stronger immunostaining and lower background stain-ing. Sections of formalin-fixed tissues are mounted on glass slides and heated in aslide oven for 1 hr at 56°C. They are deparaffinized with xylene and rehydrated withethanol to distilled water.

The slides are placed in a plastic microwavable rack and immersed in a microwavablestaining dish filled with 200 ml of 0.01 M citrate buffer (pH 6.0). Blank slides are added tothe rack to maintain a uniform volume in the container. The container is covered at anangle with the lid and heated twice for 5 min per cycle at 800 W in a microwave oven.Distilled water (50ml) is added after the first heating cycle to cool at room at room tem-perature for 10 min.

The buffer (~800 ml) is heated to boiling in the microwave oven and poured into an80-W ultrasonic cleaner (Bransonic 12, Branson Cleaning Equipment Co., Shelton, CT).The slide rack is sonicated for 1 min and then cooled in buffer in the staining container foran additional 10 min.

The sections are treated with Biotek enzyme (Ventana Biotek) for 10 min, followedby blocking of endogenous peroxidase with 3% hydrogen peroxide in buffer for ~8 min.Immunostaining is carried out in an automated immunostainer (TechMate 1000 EquipmentCo., Shelton, CT). The slide rack is sonicated for 1 min and then cooled in buffer in thestaining container for an additional 10 min.

A cocktail (1:1) of two monoclonal anticyclin D1/bc l-1 antibodies were used:P2D11F11 (diluted 1:40) and 5D4 (diluted 1:100); these two antibodies can be obtained fromVecta Laboratories, Inc., Burlingame, CA, and Immunotech, Westbrook, ME, respectively.The avidin-biotin immunoperoxidase detection system employing DAB is used as the chro-mogen (Ventana Biotek). After counterstaining in dilute Mayer’s hematoxylin, the sectionsare dehydrated and mounted in Permount.

COMBINED ENZYME DIGESTION AND MICROWAVE HEATINGANTIGEN RETRIEVAL METHOD

In certain cases maximally effective antigen retrieval conditions tend to cause non-specific staining and/or background staining. This artifact can be avoided by employing acombination of mild heating and enzyme digestion. This approach can also be used formultiple immunostaining, including that of the PCNA (Ezaki, 2000).

Tissue specimens are fixed with 4% paraformaldehyde and embedded in paraffin at60°C for 1 hr. Sections thick) are mounted on gelatin-coated glass slides, deparaf-finized, and rehydrated in distilled water. They are treated with 0.005% pepsin for 15 minat 37°C, followed by heating in 0.01 M citrate buffer (pH 6.0) in a microwave oven (300W) at 80°C for 15 min. The sections are washed in distilled water for 5 min, rinsed in0.01 M PBS (pH 7.2) for 15 min, and treated with 0.3–1% to quench endogenousperoxidase activity.

They are incubated in the primary antibody (PC10, diluted 1:200 in PBS containing0.2% BSA for PCNA) for 1–2 hr at room temperature. The sections are washed five timesfor 5 min each with PBS and then incubated in the enzyme-conjugated secondary antibodyin PBS containing 1% heat-inactivated normal rat serum for 1 hr. Horseradish peroxidase

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reaction is developed for 10–20 min at room temperature in a mixture of 10 mg of DAB in30 ml of PBS and of 30% If needed, 1 ml of 1% cobalt chloride and 1 ml of1% can be added to the DAB solution to obtain a darker reaction prod-uct and increased staining intensity.

PRESSURE COOKER–EDTA–ASSISTED ANTIGEN RETRIEVAL

Immunohistochemical staining of tonsillitis, gastric adenocarcinomas, and breast car-cinomas can be obtained using MIB-1 antibody in conjunction with EDTA-NAOH solu-tion and a pressure cooker (Kim et al., 1999b). EDTA solution is thought to be moreeffective than other buffers in unmasking the epitopes, presumably because it removes(chelates) tissue-bound calcium ions.

Deparaffinized and rehydrated tissue sections on slides are immersed in jars contain-ing 1 mM EDTA-NaOH (pH 8.0), and the jars are placed in boiling distilled waterin a stainless steel 6-liter-capacity pressure cooker with an operating pressure of103 kPa/15 psi. The pressure cooker is sealed and brought to full pressure; the duration ofheating is ~3 min. The cooker is depressurized and cooled under running tap water for~20 min.

The sections are treated with and then incubated in the primary antibody at adilution of 1:50. This is followed by sequential incubation in the biotinylated antimouseantibody and streptavidin-biotin-labeled complex. DAB is used for 5 min as the chro-mogen, and the sections are lightly counterstained with hematoxylin. Positive controlsinvolve the use of the tissue known to express the antigen under study. Negative controlsinvolve the replacement of the primary antibody with the diluent alone or with a non-immune serum.

Note: A 1 mM EDTA solution is easier to set at pH 8.0 when it is buffered. A sodiumor potassium phosphate buffer is suitable at 0.005 mM provided the grade of the reagentsis of analytical quality, i.e., the content of divalent metals is typically 0.005% or less.

2-MERCAPTOETHANOL–SODIUMIODOACETATE–ASSISTEDANTIGEN RETRIEVAL

Antigen unmasking on sections of paraffin-embedded tissues can be accomplished byreduction of disulfide bonds by treatment with 2-mercaptoethanol, followed by alkylationwith sodium iodoacetate to prevent the bonds from reforming. This method has been usedfor unmasking a Kunitz protease inhibitory domain epitope of Alzheimer’s amyloid pre-cursor protein in human brain (Campbell et al., 1999). Sections are reduced with a mixtureof 0.14 M 2-mercaptoethanol in 0.5 M Tris-HCl (pH 8.0) and 1 mM EDTA for 3 hr in thedark at room temperature. After being washed for 3 min in distilled water, the sections aretreated with a mixture of 250 mg/ml iodoacetic acid in 0.1 M NaOH, diluted 1:10 in 0.5 MTris-HCl (pH 8.0) and 1 mM EDTA for 20 min in the dark.

The unmasking of antigen with the above method can be enhanced by microwavingthe sections for 7 min in 0.05 Tris-HCl (pH 7.0). Controls include preabsorption of the

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primary antibody with the antigen and substitution of the primary antibody with non-immune serum from the same species.

Endogenous peroxidase activity is quenched by treatment with 0.5% in methanol,immunostaining is carried out using a Vectastain Elite ABC kit, and visualization of thesecondary antibody is achieved with DAB enhanced with nickel (Vector Laboratories).

ANTIGEN RETRIEVAL WITH STEAM–EDTA–PROTEASE METHOD

Basal cell–specific monoclonal antibody to high-molecular-weight cytokeratinsis routinely used to distinguish benign from malignant prostatic acini (Brawer et al., 1985).This antibody provides evidence of the absence of basal cells in prostatic cancer. However,its variable staining tendency under standard preparatory procedures is not uncommon. In thepast, antigen retrieval methods used with this antibody consisted of proteolytic digestion,microwave heating, EDTA, or urea treatment. Recently, a combined steam-EDTA-proteaseprotocol was employed in conjunction with this antibody for enhancing basal cell immunore-activity in noncancerous prostatic epithelium (Iczkowski et al., 1999). This protocol helps toprevent misinterpretation of histological mimics of cancer by improving immunohistochem-ical basal cell–specific keratin expression in benign prostatic acini and in prostatic intraep-ithelial neoplasia. The method increases the percentage and intensity of immunoreactivity inbasal cells of benign, atrophic, and hyperplastic acini without introducing background stain-ing, thus improving the diagnostic potential of cytokeratin

Procedure

Sections of formalin-fixed and paraffin-embedded tissues are placed onto silane-coated slides, deparaffinized, and rehydrated. They are placed in 0.1 M EDTA (pH 8.0) andexposed to steam heat for 30 min. The slides are cooled for 5 min, rinsed in tap water, loadedonto the ES Autostainer, and treated with Protease 2 (Ventana) for 8 min. Incubation is car-ried out in the primary antikeratin (diluted 1:10 in PBS) for 32 min and in biotiny-lated secondary antibodies followed by streptavidin for 8 min each. The staining isvisualized on the instrument using 3-amino-9-ethylcarbazole. Between each step, the slidesare rinsed in Tris-buffered saline. The results of this protocol are shown in Figure 8.8.

PICRIC ACID–STEAM AUTOCLAVING–FORMIC ACID–GUANIDINETHIOCYANATE–ASSISTED RETRIEVAL OF PRION PROTEIN

In humans Creutzfeldt-Jakob disease (CJD) is the most common of transmissible spongi-form encephalopathies (TSEs), a group of neurodegenerative diseases. The cause of theTSEs is the prion protein. A number of immunohistochemical methods are available fordetecting prion depositions in the brains of humans suffering from CJD (Kitamoto et al.,1985; Haywood et al., 1994). However, the retrieval of prion in the tissues fixed withformalin and embedded in paraffin is difficult. Hydrated autoclaving instead of microwaveheating is necessary for the retrieval of prion protein. The following sequential

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protocol—saturated picric acid–steam autoclaving–formic acid–guanidine thiocyanate—iseffective in retrieving this protein (Van Everbroeck et al., 1999).

Monoclonal antibodies 3F4 (Senetk, St. Louis, MO) and F89/160.1.5 are used forimmunostaining of prion protein. The monoclonal antibody 3F4 was developed byKascsack et al. (1987) and shows immunoreactivity with the epitope around AA 112 of thehuman prion protein. The monoclonal antibody F89/160.1.5 was developed at the U.S.Department of Agriculture against a synthetic peptide representing residues 146–159 of

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the bovine prion protein. The epitope has been mapped to the sequence IHFG. This epi-tope is also conserved in the human prion protein sequence located at AA 139–142. Thisantibody shows stronger sensitivity and specificity than those obtained with 3F4 antibodyfor the human prion protein (Van Everbroeck et al., 1999). The antibody F89/160.1.5 isused at a dilution of 1:20,000 with TBSB (150 mM NaCl, 1% BSA, 50 mM Tris-HClbuffer; pH 7.4) (final concentration is 55 ng/ml). The antibody 3F4 is used at a dilution of1:2,000 with TBSB (final concentration is 1:25 mg/ml).

Procedure

Brain tissue specimens are treated with 98% formic acid for 1 hr to reduce infectivityand embedded in paraffin. Sections thick) are picked up on 0.1% poly-L-lysine-coatedglass slides; Superfrost Plus slides are not recommended because sections tend to be dis-lodged during multiple treatments. The sections on slides are deparaffinized with xylene andrehydrated with descending concentrations of ethanol. They are treated with 5% picric acidfor 15 min at room temperature and then thoroughly washed with tap water. The sections areexposed to 0.3% in methanol for 30 min to block endogenous peroxidase. This is fol-lowed by autoclaving for 10 min at 121°C using 10 mM citric acid (pH 6.0) as the recoverybuffer. The sections are allowed to cool and then washed in distilled water. The sections aretreated with 88% formic acid for 5 min and then washed in distilled water. Finally, precooled(4°C) guanidine thiocyanate (4M) is pipetted onto the sections and incubated for 2 hr at 4°C.

The sections are exposed to normal swine serum (1:25) in TBSB (150 mM NaCl, 1%BSA, 50 mM Tris-HCl; pH 7.4) for 30 min to block nonspecific binding sites. They areincubated overnight in a humid chamber at room temperature in the primary monclonalantibody (3F4) (Senetk, St. Louis, MO) and diluted 1:2000 with the buffer. The avidin-biotin complex (ABC) method is used to detect antibody binding. The bound antibody isdetected by incubation with the secondary antibody (biotinylated goat antimouse IgGdiluted 1:100 in TBSB) for 30 min at room temperature. This is followed by incubation for1 hr with avidin-biotin-horseradish peroxidase complex, diluted 1:200 in TBSB. As thestaining mixture, 0.05% DAB in TBS (150 mM NaCl, 50 mM Tris-HCl; pH 7.6) with0.002% is used for 5 min. After thorough washing for 10 min in running tap water,the sections are counterstained with Harris’ hematoxylin for 30 sec. They are dehydratedand mounted. The results of this procedure are shown in Figure 8.9.

Great care should be taken in handling the tissue to avoid infection. Picric acid is bothtoxic and explosive. Safety guidelines must be used when working with this reagent.Guanidine thiocyanate is also a biohazardous material.

SIMULTANEOUS DETECTION OF MULTIPLE ANTIGENS

Simultaneous detection of multiple antigens provides a spatial relationship betweenthe antigens of interest and saves time, effort, and tissue specimens. In the standardimmunoperoxidase technique, horseradish peroxidase is used to oxidize the colorlesschromogen DAB into a brown end-product in the presence of hydrogen peroxide. Whennickel chloride is included in the reaction mixture, the final reaction product is black. By

General Methods of Antigen Retrieval 195

employing these two reagents sequentially, two nonoverlapping antigens can be localized,one exhibiting brown color and the other black.

Simultaneous detection of three antigens within one tissue section became possibleby employing an additional peroxidase substrate such as the Vector VIP Substrate kit(Vector Lab, Burlingham, CA) (Pujic et al., 1998). This substrate is oxidized by horseradishperoxidase and yields a rose-colored final reaction product which differs in color from that

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of the DAB-based reaction products. The sequence of application is: nickel-enhancedDAB, DAB, and VIP. Three monoclonal antibodies are applied in sequential immunoper-oxidase staining steps, resulting in the deposition of black, brown, and rose stains.Colocalization of mast cell tryptase, neurofilament protein, and CD31 in normal humanskin was accomplished using this method (Pujic et al., 1998).

Procedure

Human skin tissue is embedded in OCT, cryostat sections are prepared,mounted on poly-L-lysine-coated slides, and stored at –80°C. The sections are brought toroom temperature (22°C), and a grease ring is drawn around the sections to limit the spreadof reagents. They are rehydrated for 10 min in PBS (0.1M phosphate buffer, 0.15 M saline,pH 7.4) and treated for 15 min with blocking solution containing 2% normal swine serumand 1% BSA in PBS.

The sections are incubated for 1 hr with the primary monoclonal antibody, mouse anti-human mast cell tryptase antibody (DAKO), diluted 1:200 with 1% BSA/PBS. They arewashed for 10 min in PBS using magnetic stirring, incubated with biotinylated antimouseantibody for 15 min, and washed in PBS. This is followed by adding avidin-biotin-horse-radish peroxidase for 15 min. A Vector DAB Substrate kit is applied to develop the reactionproduct by using nickel-DAB (5 min developing time) according to the manufacturer'sinstructions. This step yields a black reaction product at sites of mast cell tryptase.

The sections are washed in PBS and treated for 15 min with 0.3% toquench residual peroxidase activity. Sections are then treated for 15 min with the blockingsolution as described above. The primary monoclonal antibody, mouse antineurofilamentprotein antibody (DAKO), diluted 1:200 with 1% BSA/PBS, is added for 1 hr. They arewashed with PBS and processed through steps with biotinylated secondary antibody andhorseradish peroxidase as outlined above. The peroxidase is visualized using a Vector DABSubstrate kit with DAB for a 5-min reaction time to yield a brown reaction product for neu-rofilaments.

Prior to the third staining sequence, the sections are washed in PBS and treated withquenching solution as described above. The primary monoclonal antibody, mouse anti-human CD31, diluted 1:200 with 1% BSA/PBS, is added for 1 hr, followed by washing inPBS. The sections are processed through steps with biotinylated secondary antibody andhorseradish peroxidase as described above. The peroxidase enzyme is developed by usinga Vector VIP Substrate kit for 4 min to yield a rose-colored reaction product on labeledendothelium. The sections are washed in PBS, counterstained lightly in Mayer’s acidhematoxylin for 20 sec, and rinsed in tap water before being dehydrated in ethanol, clearedin xylene, and mounted.

USE OF MULTIPLE ANTIBODIES FOR LABELING ANTIGENS

Immunohistochemistry of multiple antigens in monolayer cell cultures during the sameexperimental conditions is also important in cell biology. Advanced techniques for antibodyproduction combined with sensitive detection systems have facilitated the localization of

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picogram quantities of specific antigens (Zijlstra and Schelling, 1999). Two conventionalmethods for detecting multiple antigens are (1) individual antibodies against different antigensused for separate cell structures and (2) multiple primary antibodies applied against differentantigens in the same cell culture and visualized with separate staining methods. Zijlstra andSchelling (1999) have developed the following simple procedure for detecting and quantify-ing four individual fibronectin isoforms within a single fibroblast monolayer culture.

Procedure

Cells are cultured in 100-mm (internal diameter) tissue culture dishes, rinsed threetimes at 37°C with PBS, and fixed with 100% methanol for 20 min at –20°C. Themethanol is vacuum-aspirated, and residual methanol is allowed to evaporate. A grid(55 X 60 mm) is formed by placing the culture dish on top of a template and tracing thelines using a PAP-pen (the Binding Site, San Diego, CA). Thus, the surface of the dish isdivided into 20 distinct areas (11 X 15 mm). The spacing of the area in these grids allowsthe use of a multichannel pipette. The PAP-pen lines provide a hydrophobic barrierbetween each area and prevent horizontal movement of fluids.

The culture is incubated in methanol containing 3% for 10 min to quenchendogenous peroxidase activity. The cells are rehydrated in 70% ethanol for 2 min, fol-lowed by rinsing for 5 min in PBS. Nonspecific binding is blocked by incubating the cellsfor 1 hr in the blocking buffer (1% BSA in PBS).

The four monoclonal antibodies used and their specificity are shown in Table 8.1.Approximately of the primary antibodies, appropriately diluted in blocking buffer,are applied to individual areas of the monolayer. The culture dish is placed inside a humidchamber and incubated for 2 hr at room temperature. The template of the grid is used todetermine the location, quantity, and dilution of each antibody. Each area is rinsed with

of PBS, followed by three changes for 5 min each with blocking buffer AHRP-conjugated secondary antibody, diluted in blocking buffer, is applied to each area for1 hr in a humid chamber. The areas are rinsed with of blocking buffer. Approximately

of DAB is applied for 5 min, rinsed twice with distilled water, and counterstainedwith Erlich’s hematoxylin. All washing and blocking solutions are applied with a multi-channel pipette and removed by aspiration using a 1-ml syringe attached to a vacuum trap.The immunostaining is documented with a Sony DKC-5000 digital photosystem. In theabove method, antigen retrieval is not required.

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ANTIGEN RETRIEVAL IN NEURONAL TISSUE SLICES BEFOREVIBRATOME SECTIONING

Microwave heating causes severe wrinkling and folding of free-floating thickvibratome sections (Evers and Uylings, 1994b). To avoid this problem, microwaveheating is applied to slices (5 mm thick) of formaldehyde-fixed tissue before vibratome cut-ting. The following method is useful for antigen retrieval in nonembedded tissue slices fromwhich vibratome sections are prepared for immunohistochemical staining. The followingprotocol was employed for antigen retrieval in human brain tissues stored in 4% bufferedformaldehyde for as long as 4 years (Evers and Uylings, 1997; Evers et al., 1998).

A slice ~5 mm thick is cut from fixed brain tissue and washed for several hours indistilled water to remove excess formaldehyde. The slice is immersed overnight in Tris-buffered saline (TBS, pH 9.0) and then placed in a plastic jar containing ~200 ml of TBS.The jar is placed in a microwave oven for 10–15 min at full power (700 W), divided intotwo cycles of 5 or 7.5 min to check the fluid level. The temperature is controlled using thetemperature probe of the oven. It takes ~3 min to reach a temperature of 90°C.

The jar is removed from the oven and allowed to cool for 15 min. The slice is rinsedin TBS (pH 7.6), and vibratome sections are cut, collected in plastic vials, andwashed for 1 hr in TBS (pH 7.6). (These sections are relatively thick in order to stain wholeneurons, including neuronal processes, to distinguish different morphological types.) Thesections are immersed in TBS containing 3% hydrogen peroxide and 0.2% Triton X-100for 30 min to prevent endogenous peroxidase activity. Following a thorough wash in TBS,the sections are placed in TBS containing 5% nonfat dry milk and 0.2% Triton X-100 for1 hr to prevent nonspecific antibody binding to the tissue proteins.

The sections are incubated overnight in the primary monoclonal antibody in a coldroom at 4°C. These antibodies (MAP-2, SMI-32, SMI-311, SMI-312, calbindin, and par-valbumin) are diluted 1:1000 to 1:4000 with TBS containing 5% nonfat dried milk and0.2% Triton X-100. They are thoroughly washed in TBS and then incubated for 1 hr in thesecondary antibody. For monoclonal antibodies raised in mice, peroxidase-conjugated rab-bit antimouse secondary antibodies can be used. For polyclonal antibodies against calre-tinin and neuropeptide Y, which are raised in rabbits, goat antirabbit secondary antibodiescan be used. After washing in TBS, a tertiary antibody, peroxidase (PAP, 1:1000) can beused. The chromogen used is 0.05% DAB enhanced with 2% nickel-ammonium sulfate.

MICROWAVE HEAT–ASSISTED ANTIGEN RETRIEVAL IN FRESHLYFROZEN BRAIN TISSUE

The use of freshly frozen brain tissues in immunohistochemical studies has certaindrawbacks, such as poor preservation of tissue morphology and antigenicity resulting fromice crystal formation. Many antibodies available for immunohistochemistry have not beenapplied to this type of tissue. Microwave heating at boiling temperature is effective in therapid retrieval and immunostaining of antigens in freshly frozen tissues, including neuraltissues. This technique has been used to enhance staining of the glial fibrillary acidic pro-tein (GFAP) in rat brain tissue using monoclonal anti-GFAP antibody (DeHart et al.,1996). Uniform increased staining of cell bodies and large astrocytic processes occurs in

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both grey and white matter without excessive background staining. If nonuniform stainingis observed, it may be due to uneven section thickness or unevenly frozen tissues.

Procedure

Fresh rat brain tissue is immediately immersed in 2-methyl butane at –15 to –25°Cfor several minutes and stored at –70°C. Sections are cut in the parasagittal planeon a cryostat, which are thaw-mounted onto poly-L-lysine-coated glass slides and stored at–70°C. The sections are dried on a slide warmer at the lowest setting to avoid excessivedrying. They are fixed with 4% formaldehyde in PBS (pH 7.5) for 30 min and then rinsedthree times for 10 min each in PBS to remove excess fixative. This is followed by treatmentwith 0.3% hydrogen peroxide in PBS for 30–40 min to quench endogenous peroxidaseactivity.

After being washed in three changes of 5 min each in PBS, the slide is placed in aplastic jar containing 60ml of 10 mM sodium citrate buffer (pH 6.0) and 0.04% TritonX-100. The jar is loosely covered with its screw cap and heated for 5 min in a microwaveoven at high power. The buffer starts to boil after ~90 sec. The heating process is inter-rupted at intervals of 1 min so that the fluid level can be checked and replenished in the jar.The sections must not be allowed to dry. The jar is removed from the oven and allowed tocool to room temperature for 20–30 min. The sections are rinsed in three changes of 5 mineach in PBS.

The sections are treated with 10% normal horse serum for 3 hr at 4°C to reduce non-specific binding. After being rinsed three times for 5 min each in PBS, the sections areincubated in the monoclonal anti-GFAP antibody and diluted 1:400 in PBS for 18 hr at4°C. Following washing three times for 10 min each in PBS, the sections are incubated inbiotinylated secondary antibody diluted 1:200 in PBS for 1 hr at room temperature. Theperoxidase bridge is completed by treating the sections with an avidin-biotin peroxidasecomplex solution for 30 min at room temperature. The sections are rinsed twice in PBS,and immunoreactivity is visualized using DAB (10 mg/15 ml) and 0.024% hydrogenperoxide in 50 mM Tris buffer, pH 7.6). After a rinse in distilled water, the sections aredehydrated and cover-slipped.

MICROWAVE HEAT–ASSISTED RAPID IMMUNOSTAININGOF FROZEN SECTIONS

Intraoperative diagnosis requires rapid immunostaining of cryosections. Rapidimmunostaining is also helpful in confirming or excluding tumor clearance in resectionmargins or in detecting micrometastases in sentinel lymph nodes in breast cancer patients.The enhanced polymer one-step staining (EPOS) system allows a rapid one-step immunos-taining that can be completed in ~12 min. The EPOS procedure is based on the chemicallinking of primary antibodies and horseradish peroxidase to an inert polymer complex(dextran) (Bisgaad et al., 1993). This methodology has been employed for immunostain-ing of Ki-67, PCNA, cytokeratin, and leukocyte common antigen (Tsutsumi et al., 1995;Richter et al., 1999).

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Procedure

Sections ( thick) of freshly frozen tissues are mounted on silane-coated slidesand fixed with 4% buffered formaldehyde (pH 7.0) for 20 sec (Richter et al., 1999). Thesections are rinsed in TBS (pH 7.4) for 15 sec, followed by incubation with EPOS antibodyfor 3 min at 37°C in an incubation chamber. They are rinsed twice for 15 sec each inTBS and then developed with peroxidase-DAB detection kit (Dako) in a microwave oven(500 W) for ~ l min; during microwaving, the slides are cooled by a cold water bath(Werner et al., 1991). After being rinsed in tap water, the sections are counterstained withhematoxylin for 10 sec. They are rinsed in tap water and cover-slipped.

MICROWAVE HEAT–ASSISTED IMMUNOCYTOCHEMISTRYOF THIN CRYOSECTIONS

As is true of formaldehyde-fixed and paraffin-embedded tissue sections for lightmicroscopy, thin cryosections of aldehyde-fixed tissues for electron microscopy also showimproved labeling efficiency with microwave heating in some systems. Cryosections canbe heated in a microwave oven prior to antibody application. Heating diluted antibodiesbefore their application may also result in improved labeling, but generally this is not true.Typically, protein solutions lose efficiency in the microwave oven. However, the labelingof all types of antigens on cryosections is not improved with microwave heating. Also, thelabeling efficiency is affected by the type of fixation and the heating duration.

It was demonstrated that optimal labeling, for example, of amylase in thin cryosec-tions of formaldehyde-fixed rat pancreas tissue occurred with microwave heating at 25°Cand full power for 2 min (Chicoine and Webster, 1998). In contrast, this duration of heat-ing had no effect on similar sections of the same tissue fixed with glutaraldehyde. On theother hand, thin cryosections of glutaraldehyde-fixed tissues showed increased labelingafter microwave heating for 6 min. The duration of heating producing the highest specificsignal density in the glutaraldehyde-fixed tissues tends to exert an adverse effect on thesignal density of the formaldehyde-fixed tissues.

Longer durations of heating are required for the glutaraldehyde-fixed tissue sectionsbecause this dialdehyde compared with monoaldehyde formaldehyde introduces moreextensive and stronger protein crosslinks. The presence of strong protein crosslinks is abarrier to the accessibility of the antigens to the antibody. Therefore, longer heating isrequired to break down the protein crosslinks produced by glutaraldehyde.

Several possible explanations can be offered to clarify the increased labeling effi-ciency of antibodies, which may occur because of easy penetration of antibodies into thincryosections. Antigens are thought to be exposed more readily on cryosections because ofthe absence of chemical treatments, such as resin embedding and alcohols, after the initialfixation step. Heating of thin cryosections may stabilize the antigens. Cryosections thathave been cut from tissue blocks which have been fixed and infiltrated with sucrose are notsubjected to additional processing steps (e.g., dehydration with alcohols and embedding inresin), which may wash away soluble antigens such as amylase (Chicoine and Webster,1998). Heating of thin cyrosections may also unpack the antigen from the surrounding pro-teins and/or unfold the antigen molecule, facilitating epitope accessibility to the antibody.

General Methods of Antigen Retrieval 201

It is known that antibody labeling efficiency over tightly packed antigens is reducedbecause of steric hindrance.

Procedure

Small pieces of tissue are fixed for 1 hr in a mixture of 4% formaldehyde and 0.5%glutaraldehyde in 100 mM phosphate buffer (pH 7.4) (Chicoine and Webster, 1998). Theyare infiltrated with 2.3 M sucrose for 24 hr at 4°C, mounted with specimen pins (Leica,Deerfield, IL), and frozen by immersion in liquid nitrogen. The frozen tissue is sectionedat –110°C using an Ultracut E ultramicrotome (Leica) equipped with a diamond knife andan FC4 cryoattachment. Cryosections ~60–80 nm thick are thawed and then mounted onFormvar-carbon-coated grids.

A microwave oven equipped with Pello 3420 Load Cooler attachment is set at 25°Cand full power. Two beakers, each filled with 500 ml of water, are placed in previouslydetermined hot spots in the microwave oven (see pages 102–103). Grids containing thethin cryosections are floated (section side down) sequentially on small drops of 0.15%glycine and 1% BSA for 15 sec and 5 min, respectively. The reagent drops are placed on aclean disposable plastic surface which has been placed on the cold spot in the oven (an areabetween the two beakers of water). The local temperature is controlled using themicrowave temperature probe immersed in a tube of water placed close to the grids.

The sections are floated on small drops of appropriately diluted primary antibody andintermittently microwaved for a total duration of 8 min: 2 min with microwave oven on,2 min with microwave oven off, 2 min with oven on, and 2 min with oven off. This is fol-lowed by washing twice with PBS for 15 sec in a microwave oven. The sections are treatedoutside the oven for 15 min with protein A–colloidal gold (10 nm) complex diluted 1:20 to1:50 (determined by spectrophotometry) in PBS containing 1% BSA. They are washed inPBS followed by distilled water and counterstained outside the oven.

Alternatively, the sections are microwaved for 6 min before incubation with the pri-mary antibody. The remaining steps are also carried out outside the microwave oven.

PRESSURE COOKER–ASSISTED DETECTION OF APOPTOTIC CELLS

To detect apoptosis in paraffin tissue sections, the TUNEL technique is most com-monly used. However, this technique is not specific because it also detects nonspecificDNA degradation in autolysis or necrosis, and DNA breaks during DNA repair, resultingin false-positive staining (Suurmeijer et al., 1999). Alternatively, the apoptotic pheno-type can be visualized by immunostaining target proteins cleaved by caspases, becausethe process of apoptosis is irreversible once the caspase cascade is completelyactivated. Immunodetection of caspase-cleaved cytokeratin 18 has been accomplished(Caulin et al., 1997). However, this protocol is specific for detecting cytokeratin18 in apoptotic cells in epithelial tissues or tumors, although immunostaining of cleavedactin filaments is more useful because of its omnipresence in human apoptotic cells.However, whether actin cleavage by caspases is a universal mechanism in apoptosis hasnot been established as yet.

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A polyclonal antibody (fractin) is specific for an actin fragment generated bycaspases during apoptosis. This antibody does not react with intact actin filaments. It isobtained by injecting rabbits with a synthetic peptide (K-YELD) representing the last fiveamino acids of the C-terminus of the 32-kDa actin fragment produced during apoptosis,

residues 240–245 (YELPD) coupled via the K-residue to a carrier protein (purifiedprotein derivative of tuberculin) (Yang et al., 1998). Stock solutions of the antibody areprepared by resuspending of lyophilized rabbit antiserum in distilled water.

Suurmeijer et al. (1999) tested three antigen retrieval methods using fractin antibody:microwave heating with 10 mM citrate buffer (pH 6.0), pressure cooking with 1 mMEDTA (pH 8.0), and overnight heating at 70°C with Tris-HCl buffer (pH 9.0). The pres-sure cooker method yielded the most consistent immunostaining. Light microscopic fea-tures of apoptotic cells are nuclear shrinkage and chromatin condensation. Fragmentedapoptotic bodies show strong immunostaining (Fig. 8.10/Plate 4E).

IMMUNOHISTOCHEMICAL LOCALIZATION OFPROSTATE-SPECIFIC ANTIGEN

Prostate-specific antigen (PSA) is a chymotrypsinlike serine protease synthesizedprimarily by the normal, hyperplastic, and malignant male and female prostate. In the maleits expression is tightly regulated by androgen through the action of androgen receptor(AR). Upon binding to androgen, AR translocates it into the nucleus and binds to theandrogen response elements (AREs) on the PSA promotor, where it interacts with othertranscription factors and activates PSA gene transcription.

Prostate-specific antigen is currently the most frequently used marker for identifyingnormal and pathologically altered prostatic tissue in the male and female. At present, there

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is no reason to use the terms Skene’s gland and/or paraurethral ducts and glands for thehuman female prostate 1999, and Ablin, 2000).

Immunohistochemical studies demonstrate that PSA is expressed in the highly special-ized, apically superficial layer of male and female secretory cells of the prostate gland, as wellas in uroepithelial cells at other sites of the urogenital tract of both sexes. Such studies pro-vide evidence that PSA plays a crucial role in the identification of normal or pathologicallyaltered prostate tissue. In clinical practice, PSA is a valuable marker for diagnosing and mon-itoring prostate cancer in both sexes. and Ablin (2000) have reviewed the functional-morphological and some clinical aspects of the normal and pathological female prostate.

Prostate-specific antigen is also a serum marker for prostate cancer. The serum PSAis generally proportional to tumor volume and correlates positively with the clinical stageof the disease. Progression of prostate cancer to androgen independence is commonlyassociated with a rebound of serum PSA. Prostate-specific antigen elevation in hormone-refractory prostate tumors is attributed to mutations and/or amplifications of AR, whichbroaden its ligand specificity and/or enhance tumor cell’s responsiveness to androgen,respectively. Hormone-refractory prostate cancer is one of the most detrimental diseasesaffecting men in the United States. In males a range of 1–2 ng/ml in serum is normal in themajority of cases, while values above 3–4 ng/ml are indicative of prostate cancer, benignprostate hyperplasia, or prostatitis. It is noted, however, that exceptions do occur and thatup to 40% of individuals with levels <4 ng/ml may have prostate cancer.

A healthy female with a normal prostate is characterized by a broad range of serumPSA values from practically unappreciable amounts to the highest reported ones of0.9 ng/ml. This value is very close to the normal reference range in the male andAblin, 2000).

IMMUNOHISTOCHEMISTRY

An immunohistochemical examination of PSA using polyclonal antibodies by the per-oxidase antiperoxidase (PAP) method and by the technique of biotin-streptavidin-alkalinephosphatase has been successfully carried out et al., 1994). Immunoelectronmicroscopy in conjunction with the protein A–gold complex can also be used for localizingPSA in human prostate (Sinha et al., 1987). The procedure for immunofluorescence local-ization of PSA is given below.

Procedure

Paraffin-embedded prostatic tissue sections thick) are deparaffinized and rehy-drated (Scorilas et al., 2000). A 5% universal tissue conditioner (Biomeda) is applied for10 min at room temperature to block any nonspecific binding. The sections are incubatedwith biotinylated monoclonal mouse antihuman PSA antibody (~2mg/l) (DiagnosticSystems Laboratories [coded 8301]) for 1 hr at 37°C. The sections are stained with

for 25 min at 37°C. After each step, the sections are rinsedbriefly with 0.5 ml/1 Tween 20 solution. The slides are dried with a stream of cold air, andthe resultant fluorescence is observed with a time-resolved fluorescence microscope.

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Other Applications of MicrowaveHeating

CARBOHYDRATE ANTIGENS

Molecules other than proteins are also antigenic to various degrees. However, proteinsare the most effective antigens because of their size and structural complexity, and almostall proteins larger than 1kDa are antigenic. Complex carbohydrates, especially thosebound to proteins or lipids (e.g., glycoproteins and glycolipids) are also of immunologicalimportance. Simple polysaccharides, such as starch and glycogen, are usually not effectiveantigens because they are rapidly degraded within cells and, in addition, do not formstructurally stable epitopes.

Lipids are poor antigens because of their wide distribution, relative simplicity, structuralinstability, and rapid metabolism. However, when linked to proteins or polysaccharides theymay function as haptens. Nucleic acids are also poor antigens because of their relative sim-plicity and flexibility and rapid degradation. Nevertheless, antibodies against DNA and RNAcan be produced by stabilizing and linking them to an immunogenic carrier. In fact, severalserious human autoimmune diseases, such as systemic lupus erythematosus, are the result ofautoantibodies to nucleic acids. The discussion below is limited to carbohydrate antigens.

Correlation between molecular alterations and tumor behavior is well established.Tumor behavior is related to the products of changes in cancer-related genes. The study ofindirect gene products such as cell surface carbohydrates is important in understandingmalignancy because tumor development is usually associated with changes in thesecarbohydrates. These changes are often divided into alterations related to terminal carbo-hydrate structures (which include incomplete synthesis and modification of normally exist-ing carbohydrates) and alterations in the carbohydrate core structure. The latter includeschain elongation of both glycolipids and proteins, increased branching of carbohydrates inN-linked glycoproteins, and blocked synthesis of carbohydrates in O-linked mucinlikeglycoproteins. The importance of studying such changes becomes obvious considering thatthe expression of carbohydrate antigens increases in a number of epithelial malignancies,including ovary, lung, bladder, breast, colon, prostate, and gastric carcinomas. In fact,tumor-associated carbohydrate changes are being used in the diagnosis of human cancers,and the expression of some carbohydrate structures is associated with prognosis.

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Carbohydrates such as glycolipids and glycoproteins (including mucins) are exten-sively expressed on the plasma membrane. They are also present on the membranes of cellorganelles such as Golgi apparatus, nuclei, mitochondria, and lysosomes. They function asstructural components and are also involved in a variety of cellular functions such as cel-lular adhesion, recognition and signal transduction, cell matrix interactions, and cell pro-liferation. Carbohydrate antigens can serve as tumor-associated antigens in carcinomas byvirtue of their overexpression or cellular distribution and accessibility. The majority ofthese carbohydrate antigens are blood group related, belonging either to the A, B, H, orLewis family, or to the mucin core family (TF, Tn, and sialosyl-Tn).

Cellular posttranslational modifications in the maturation of some proteins in bothbenign and malignant cells require N- and O-linked glycosylation. Changes in glycosyla-tion of both glycolipids and glycoproteins in tumors are well known, and aberrant glyco-sylation in tumors and tumor-associated carbohydrate antigens has been demonstrated(Hakomori, 1989). It is also known that up-regulated expression or loss of expression ofvarious carbohydrate antigens on the surface of the plasma membrane of cancer cells isassociated with a metastatic phenotype. Moreover, such alterations are related to poorpatient survival in a number of epithelial malignancies.

Carbohydrate antigens are expressed in many organs and tissues, including ovaries;other examples are given below. Sialosyl-Tn antigen is expressed in most of the pancreaticcarcinomas, whereas it is completely absent in the normal pancreas (Osaka et al., 1993). BothTn and sialosyl-Tn antigens have been reported to be present at a higher rate in colorectalcarcinomas than in normal colorectal mucosa (Orntoft et al., 1990). Up-regulation of sialyl-

is considered a prognostic parameter in metastatic prostrate cancer (Jørgensen et al.,1995). Sialosyl-Tn antigen expression occurs early in human mammary carcinogenesis and isassociated with high nuclear grade and aneuploidy (Cho et al., 1994). Limited expression ofsialosyl-Tn has been detected in nonmalignant glandular tissues (e.g., mucinous salivarygland cells, goblet cells of the small intestine and bronchus [Yonezawa et al., 1992;Therkildsen et al., 1994]). However, according to Zimmerman et al. (1999), sialosyl-Tn, as anisolated detection factor, lacks sufficient sensitivity to be of diagnostic value in discriminat-ing malignant from benign mesothelium in body cavity effusions. It should also be noted thatsialosyl-Tn expression in a variety of carcinomas is not uniformly detected. Because ovariancarcinoma is the leading cause of death from gynecological cancers, and has been investigatedextensively, the involvement of carbohydrate antigens in this cancer is summarized below.

Ovarian Carcinoma

Ovarian epithelial tumors, the most common ovarian malignancy, are usually categorizedinto four main types: serous, mucinous, endometrioid, and clear cell. The tumors are fur-ther classified as benign, of low malignant potential (borderline), or malignant, which arefurther differentiated into four grades. The histological patterns, especially of malignanttumors, may be a mixture of varying proportions of different histological types. Ovariancarcinoma is often asymptomatic in its early stages. Approximately two-thirds of thepatients are diagnosed with stage III and IV disease, when metastatic spread is presentwithin the peritoneal cavity and/or to distant organs. Serous carcinoma is the most seriousand common histological subtype of ovarian carcinoma.

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Monoclonal antibodies are available to identify various types of ovarian epithelialtumors through immunohistochemically detecting specific antigens. Antigenic specifici-ties belonging to the O(H) and Lewis blood group families (H-l, H-2,

and or the mucin-core family (Tn, sialosyl-Tn and TF) have beenstudied by using 12 antibodies (Federici et al., 1999). A distinct difference in antigenexpression between mucinous and other histological types of ovarian carcinomas (serousand endometrioid) is found. The majority of mucinous tumors express sialosyl-Tn, and

antigens strongly and relatively homogeneously, whereas serous and endometri-oid tumors rarely express these specificities. In contrast, the latter tumors strongly express

and H type 2 antigens. However, according to Tashiro et al. (1994), simultaneousexpression of sialosyl-Tn and Tn is observed in both mucinous and serous carcinomas.Such a simultaneous expression is closely connected with malignant change. Sialosyl-Tnand Tn antigens are not expressed in normal ovarian tissues, except for sialosyl-Tn stainingof stromal capillaries.

Tn is a known cancer-related antigen of the mucin-type polysaccharides. Its presence isthe result of incomplete glycosylation. The sialosyl-Tn antigen is formed by the addition ofsialic acid to the Tn antigen by 6-sialyltransferase. Immunohistochemical studies havedemonstrated that sialosyl-Tn antigen is useful in the histological classification of ovariancarcinomas and in the determination of the malignant potential of such lesions (Ryuko et al.,1993). In contrast to the CA125 antigen, which is a useful tumor marker for nonmucinousadenocarcinomas, sialosyl-Tn is a useful tumor marker for mucinous adenocarcinomas(Kjeldsen et al., 1988). The expression of sialosyl-Tn antigen increases with the transitionfrom benign adenoma to adenocarcinoma. Monoclonal antibody TKH2 (mouse IgGl)specifically recognizes sialosyl-Tn antigen (Fig. 9. 1/Plate 4F). Like Tn antigen, immunohis-tochemistry of sialosyl-Tn is important because it is a cancer-related antigen. Sialosyl-Tn isnot influenced by blood type, menstrual cycle, menopause, pregnancy, or parturition.

Recently, Davidson et al. (2000) have evaluated the differences between carbohydrateantigen expression in primary tumors and their respective metastatic lesions, as well as the

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role of these antigens in predicting survival in advanced-stage ovarian carcinomas.This study indicates that sialosyl-Tn, and Tn antigens are widely expressedin primary ovarian carcinomas and their metastases. The diagnostic role of inner-coreO-glycans and Lewis family antigens in ovarian carcinomas also has been reported byDabelsteen (1996).

In addition to its role in ovarian cancer, sialosyl-Tn is expressed in many other organsand tissues; some examples are given below. Sialosyl-Tn antigen is expressed in most of thepancreatic carcinomas, whereas it is completely absent in the normal pancreas (Osaka et al.,1993). Both Tn and sialosyl-Tn have been reported to be present in a higher rate in col-orectal carcinomas than in normal colorectal mucosa (Orntoft et al., 1990). Up-regulationof is considered a prognostic parameter in metastatic prostate cancer (Jørgensenet al., 1995). Sialosyl-Tn antigen expression occurs early in human mammary carcinogen-esis and is associated with high nuclear grade and aneuploidy (Cho et al., 1994). Limitedexpression of sialosyl-Tn has been detected in nonmalignant glandular tissues (e.g., muci-nous salivary gland cells, goblet cells of the small intestine and bronchus) (Yonezawa et al.,1992; Therkildsen et al., 1994). According to Zimmerman et al. (1999), sialosyl-Tn, as anisolated detection factor, lacks sufficient sensitivity to be of diagnostic value in discrimi-nating malignant from benign mesothelium in body cavity effusions. It should be notedthat sialosyl-Tn expression in a variety of carcinomas is not uniformly detected.

Microwave Heat–Assisted Carbohydrate Antigen Retrieval

Sections thick) of formalin-fixed and paraffin-embedded ovarian tissue aremounted on silane-coated slides and air-dried for 24hr at 3°C (Davidson et al., 2000). Theyare deparaffinized, rehydrated, placed in 0.01 M sodium citrate buffer (pH 6.0), and heatedtwice for 5 min each in a microwave oven. 2H5 antibody (PharMingen, Becton Dickinson,San Jose, CA) is used at a concentration of to detect sialyl antigen. Stainingis performed with labeled avidin-biotin. Negative controls consist of the exclusion of theprimary antibody, while positive controls consist of carcinomas that have been shown tobe immunoreactive for the antigen in earlier studies.

Enzyme Digestion–Assisted Carbohydrate Antigen Retrieval

Both protein antigens and carbohydrate antigens can be retrieved in formalin-fixed andparaffin-embedded tissues using enzyme digestion pretreatment, although the former gen-erally are better retrieved by heat pretreatment. Guhl et al. (1998) have achieved enhancedspecificity and intensity of immunogold labeling of sugar moieties (poly a 2, 8 KDNglycotope of megalin) present on O-glycosidically linked oligosaccharides by pretreatingthe sections with N-glycanase F. This treatment also augments immunogold labeling ofcertain membrane proteins in thin cryosections at pH 5 to 6.

The mechanism responsible for improved detection is thought to be better accessibil-ity of glycotopes to the antibody; glycotopes usually are masked by unrelated largeoligosaccharides. The enzyme treatment eliminates steric hindrance by these oligosaccha-rides. The mechanism of antigen retrieval essentially is based on the depolymerization of

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methylene and polymethylene bridges introduced by formaldehyde fixation. These bridgesare known to depolymerize at acidic conditions (pH 5–6).

NUCLEOLAR ORGANIZER–ASSOCIATED REGION PROTEINS

The metaphase nucleolar organizer regions (NORs) are chromosomal segments or loops(rDNA) containing ribosomal genes associated with proteins such as upstream binding factorand RNA polymerase 1. These genes are clustered in 10 loci of the human acrocentric chro-mosomes 13, 14, 15, 21, and 22. The transcriptional activity of these intranuclear segmentsplays a pivotal role in the formation of nucleoli, directing the synthesis of both ribosomes andassociated proteins. The set of proteins associated with NORs are an acidic, nonhistone typethat binds silver ions and that are selectively stained. Silver binds with those rDNA sites thatare transcriptionally active or have already been transcribed and still retain residual rRNAnon-histone-associated proteins. The NORs stained with silver and the argyrophilic NOR-associated proteins are called AgNORs and AgNOR-associated proteins, respectively.

The AgNOR proteins appear as distinct black structures under the light microscope.Three different AgNOR staining patterns of metaphase chromosomes in human lympho-cytes have been identified (Héliot et al., 2000): pair, sticklike, and unstained structures.Chromosomes 13, 14, and 21 carry predominantly pair or sticklike AgNOR structures,while 15 and 22 carry mainly pair AgNOR structures or remain unstained. DifferentAgNOR shapes represent both the number of ribosomal genes carried by each chromo-some and the differential recruitment of active ribosomal genes in each NOR cluster.In interphase cells, the silver-stained structures are exclusively located within nucleoli(Fig. 9.2). At the ultrastructural level each silver-stained structure corresponds to a fibril-lar center with a closely associated dense fibrillar component (Derenzini et al., 1990).

Silver-stained NOR-associated proteins play a key role in the control of ribosomalRNA (rRNA) transcription and processing and are considered markers of active ribosomalgenes. The close relationship between the rate of cell proliferation and ribosomal biogen-esis is well established. In fact, a linear correlation exists between AgNOR counts and thegrowth fraction in various malignancies, including carcinoma of the breast (Öfner et al.,1996). For example, AgNOR parameters correlate significantly with MIB-1 growth frac-tion and p53 protein expression (Bànkfalvi et al., 1998). AgNOR expression is markedlyhigher in cycling (MIB-1 positive) tumor cells than in resting (MIB-1 negative) ones.However, the AgNOR size rather than its number may correlate positively with elevatedproliferative status. Cumulative evidence indicates that malignant cells frequently exhibitmore AgNOR protein compared with that in benign or normal cells. Pich et al. (2000) havepresented a list of 29 tumor types for which AgNOR protein quantity is a prognostic factor.

Silver staining of NORs is influenced by the fixative, temperature, and duration ofstaining. In general, the higher the temperature, the shorter the time required to stainNORs. Prolonged staining results in nonspecific staining, and if staining is excessive, thewhole nucleus may appear homogeneously stained with silver. Considering the numerousvariables mentioned above and others that may influence NOR stainability, disagreementsover interpreting staining results in different laboratories are inevitable. Therefore, stan-dardization of the preparatory protocols is necessary to obtain reproducible results. Thestandardized silver staining procedure consists of heating the sections in a microwave

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oven, an autoclave, or a pressure cooker. Section staining is carried out in the dark at 37°Cusing prewarmed solutions.

Two methods are available for the quantitative analysis of AgNOR proteins: the count-ing technique and the morphometric method (Trerè, 2000). The counting technique consistsof enumerating each silver-stained dot per cell under the light microscope at a magnificationof 100X. The limitation of this technique is that when single AgNOR dots are clusteredtogether or partially overlapped, it becomes subjective and poorly reproducible. Further-more, the counting technique does not take into consideration the size of eachsilver-stained dot, which is variable. Clustering of dots and dimensional variability of thedots are common in rapidly proliferating cancer cells. Another disadvantage of this techniqueis significant interobserver variation. In addition, the technique may fail to demonstrate anyprognostic relevance of the AgNOR number to neoplastic diseases, including colorectalcarcinoma and breast cancer (Hennigan et al., 1994; Toikkanen and Joensuu, 1993).

To circumvent the above limitations, the morphometric method can be used (Rüschoffet al., 1990). It consists of automatic or semiquantitative measurement of the area occu-pied by the silver-stained structures within the nuclear profile with computer-assistedimage analysis (Trerè et al., 1995). This method is faster, more accurate and reproducible,and shows less interobserver variation. Moreover, in contrast to the counting technique, themorphometric method is predictive of patient survival, independent of the clinical stage ofthe disease (Öfner et al., 1995a,b).

The morphometric method can be carried out with a CCD camera mounted on a lightmicroscope and connected to a personal computer equipped with specific morphometricsoftware. For example, Leica Quantimet SOOC image analyzer and processing system canbe used. A JVC TK-1280E videocamera, connected to a Leitz Orthoplan light microscope,is used to record the images. QWIN VO1.00 software (Leica) can be used. The area ofthe nucleus and of each AgNOR, the total area of AgNORs, and the area ratio of AgNORs/nucleus (AR) are calculated automatically, together with AgNOR length, breadth, perime-ter, roundness, and the aspect ratio of each nucleus (Staibano et al., 1998). Values areexpressed in micrometers.

Procedure

The following procedure is more suitable for routine application than other methods;as many as 200 specimens can be processed at a time with this procedure. Sectionsthick) of formalin-fixed and paraffin-embedded tissues are mounted on silane-coatedslides. They are deparaffinized with xylene and rehydrated in a series of descending con-centrations of ethanol. The sections are immersed in 0.01 M sodium citrate buffer (pH 6.0)in plastic Coplin jars and heated in an autoclave at 120°C for 20min. After the sectionshave cooled down to room temperature for 20 min, they are incubated in the freshly pre-pared following silver staining solution for 25 min at room temperature.

2% gelatin in 1% formic acid25% aqueous silver nitrate

1 part2 parts

The sections are thoroughly rinsed in deionized water to remove unwanted silver precipi-tates, dehydrated in a series of ascending concentrations of ethanol, cleared in xylene, and

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mounted. A second set of sections are stained without autoclave pretreatment. The resultsof this procedure are shown in Figure 9.3.

Nucleolar Size

Hypertrophy of the nucleolus is one of the most distinctive cytological features ofcancer cells. Malignant cells usually display a larger nucleolus than do benign cells,

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although nucleolar hypertrophy also occurs in normal proliferating cells. Thus, the valueof changes in nucleolar size in tumor pathology has been questioned. However, the rela-tionship between nucleolar function as well as nucleolar size and cell doubling time indi-cates the importance of the nucleolus in tumor pathology. Also, it has been shown that incancer cell lines characterized by different proliferation rates, the transcriptional activityof RNA polymerase 1 and the expression of the major nucleolar proteins involved in thecontrol of rRNA transcription and processing (e.g., RNA polymerase 1, nucleolin, fibril-larin, and protein B23) are directly related to nucleolar size and the rapidity of cell prolif-eration (Derenzini et al., 1998).

To evaluate nucleolar size, histological sections are silver-stained for AgNORproteins. This procedure facilitates clear visualization of nuclear structure and size that canbe precisely measured by computer-assisted morphometric analysis (Öfner et al., 1995a).Nucleolar size reliably indicates the rapidity of cell proliferation, inferring the prolifera-tion rate of cancer cells. Higher AgNOR protein values correspond to worse clinicaloutcomes. Fast-growing tumors have greater rRNA transcriptional activity than slowlygrowing ones (Derenzini et al., 2000). Therefore, the shorter the cell cycle, the greater isthe rRNA transcriptional activity per unit of time and the greater the nucleolar size. Thisconclusion is logical because proliferating cells must synthesize an adequate ribosomalcomplement for the daughter cells.

IN SITU HYBRIDIZATION

The in situ hybridization (ISH) technique was introduced in the late 1960s, openinga new era in histology and cell biology (Gall and Pardue, 1969). The technique was orig-inally applied for localizing specific DNA sequences on chromosomes or interphasenuclei. Interphase cytogenetics can be a very useful tool, for example, for bridging the gapbetween cell culture and histology in tumor cytogenetics. The method is also important forlocalizing specific mRNA sequences within cells and tissues. It allows the study of chro-mosomal aberrations in routinely processed tissues. The overall advantage of ISH is thatby recognizing specific DNA or RNA sequences in a tissue or a cell, the precise locationof a potential or an effective synthesis of a given molecule can be determined. On the otherhand, immunocytochemistry demonstrates only the presence of protein molecules afterthey have been synthesized.

In situ hybridization was derived from the techniques of molecular hybridization ofnucleic acids that are isolated from a particular cell population or tissue and bound to solidsupports. Hybridization of such averaged membrane-bound nucleic acids identifies differ-ent classes of DNA (Southern blot) and RNA (Northern blot). However, ISH fills the gapbetween the detection of a specific sequence and its precise location within the tissue orthe cell (Chevalier et al., 1997).

Literally hundreds of different hybridizations can be accomplished because ISH is oftencarried out using semithin or thin sections of single tissue specimen (e.g., surgical biopsy),using light and electron microscopy, respectively. A single specimen, on the other hand, isoften insufficient for Northern or Southern blot analysis. Another advantage of ISH is that itallows the establishment of libraries of paraffin- or resin-embedded or frozen tissues. It hasbeen demonstrated, for example, that the hybridization signal can be maintained in frozensections stored at –70°C with a dessicant for more than 6 years (Wilcox, 1993).

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As in the case of immunocytochemistry/antigen retrieval, a number of factors influ-ence the sensitivity and efficiency of ISH, so they must be applied optimally. The intensityof the ISH signal is affected by the type and concentration of the fixative and the durationof fixation. Dehydration and embedding procedures are also important. All of these pro-cedures must allow the retention of the target of hybridization and/or the hybridized prod-ucts. The longer the fixation, the more difficult the detection of nucleic acids. The reasonis similar to that described earlier for proteins, i.e., strong crosslinking of chromosomalDNA with nuclear proteins. A number of other factors, including the nature of the nucleicacid and its location, the type and efficiency of probe labeling, probe construction andhybridization conditions, and signal detection technique, also contribute to the success ofISH studies. Although the type of nonradioactive probe used does not affect significantlythe sensitivity of this technique, the enhancement of signal intensity is influenced by thespecific probe used.

Another relevant factor is whether enzyme digestion, detergent treatment, ormicrowave heating is used to enhance ISH detection of nucleic acids. Although enzymedigestion (proteinase K) alone is being employed for enhancing ISH detection of nucleicacids, this approach is not preferred, for it may adversely affect tissue morphology.Moreover, some tissues are resistant to enzymatic digestion. However, proteolytic pre-treatment alone is effective for mildly fixed tissues. Denaturing agents such as sodiumthiocyanate and Tween 20 have also been used, but the results are capricious and morpho-logical details are not well preserved. However, detergent treatment may have to be usedfor tissues rich in lipids. In addition, a mild detergent treatment is better for ultrastructuralpreservation and cell cultures. Microwave pretreatment is the method of choice, for itprovides homogeneous hybridization even on large tissue sections. Some evidence indi-cates that microwave heating is more effective than other types of heating. It is emphasizedthat the hybridization of labeled probes with the target nucleotides is limited by the abil-ity of the probe to enter the section and by the accessibility and orientation of the target atthe surface of the section (Chevalier et al., 1997).

In certain cases microwave heating or enzyme digestion alone is insufficient to enhancesignal detection. This problem can be avoided by using a combination of microwave heatingand enzyme digestion. It was demonstrated, for example, that in comparison with proteinaseK digestion or heating alone, short-term (1 min) microwave heating followed by enzymedigestion significantly enhanced the detection of apoptotic cells as well as the staining inten-sity of the labeled nuclei by the T&T-mediated nick end–labeling technique (TUNEL)(Sträter et al., 1995). The TUNEL method is employed for in situ detection of DNA frag-mentation and, thus, of apoptotic cells (Gavrieli et al., 1992).

Various mechanisms responsible for the effectiveness of the above-mentioned com-bined technique have been suggested. Heating may increase the accessibility of proteins toprotease attack by stimulating the diffusion and/or enhancing the reaction rates.Microwave heating is known to rapidly hydrolyze proteins and peptides. In addition,microwave pretreatment allows reduction in the effective enzyme concentration as well asduration of enzyme digestion. Proteolytic enzymes also hydrolyze proteins, rendering tar-get nucleic acid more accessible to the probe. Alternatively, microwave heating maydirectly affect the conformation of the nucleic acid molecule, facilitating denaturation ofdouble-stranded sequences into single strands or unwinding single-stranded structureswhich may have self-annealed (McQuaid et al., 1990). It has been suggested that high

Other Applications of Microwave Heating 215

temperatures may disrupt the nucleocaspid protein coat of the measles virus, exposingmore nucleic acid for in situ hybridization (Shapshak et al., 1985). Another possible expla-nation is that heating denatures the self-hybrids formed by folding of an mRNA moleculeor pseudohybrids formed between neighboring mRNA molecules in tissue sections(Sibony et al., 1995). Heating may loosen these single mRNA strands, enhancing thein situ hybridization efficiency.

The role of high temperatures in the breakdown of protein crosslinks introduced byaldehydes has been discussed earlier in this book. The aforementioned combined tech-nique is especially effective in increasing the in situ hybridization signal in archival tissues,the fixation history of which may or may not be known. This protocol also detects lowlevels of nucleic acids in the tissue, which may not be detectable with heating or enzymedigestion alone.

In some cases, the use of medium wattages (e.g., 450 W) is preferred over high-powermicrowave outputs (e.g., 700 W). This difference has been demonstrated in the ISH fordetecting measles virus and chicken anemia virus in formalin-fixed, paraffin-embeddedbrain tissue (McMahon and McQuaid, 1996). In this study higher power outputs resultedin decreased sensitivity. Although the exact reason for this phenomenon is not known, it ishypothesized that optimal oscillation of dipolar molecules produces optimal thermaleffects in tissue sections at medium wattages.

Urea (0.01 M), sodium carbonate (0.01 M), magnesium chloride (0.01 M), or distilledwater can be used as microwave fluids to obtain similar results in terms of both sensitivityand intensity of the hybridization signal. Alternatively, 10 mM citrate buffer (pH 6.0) canbe used as the microwave fluid. The major role of these fluids is to mediate high temper-ature effects, which is confirmed by the achievement of a good hybridization signal usingdistilled water. Note that pretreatment conditions must be optimized for every tissue typeand for every cell type in a given section.

Radioactive Probes

Radioactively labeled DNA and RNA probes as originally used (Gall and Pardue,1969) are still widely applied for ISH because of their high sensitivity and strong amplifi-cation of autoradiography. Also, radioactive probes enter tissue sections relatively easily.Signal detection can be achieved within weeks with probes, whileprobes yield autoradiographs within days. The disadvantages of these probes includesafety problems, reduced stability of radioactively labeled probes, and long durations ofexposure. These and other reasons stimulated interest in the development of nonradioac-tive probes such as biotin and digoxigenin discussed below.

Nonradioactive Probes

Biotin is a small vitamin molecule ( 244) that binds with high affinityto avidin. Avidin is a larger glycoprotein molecule ( 70,000), mostly distributed in eggwhites. This protein has the advantage of conjugating with different markers, includingperoxidase, fluorescent dyes, colloidal gold, and ferritin. Because of this property it is

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extensively used in immunocytochemistry to detect biotinylated molecules. However, non-specific binding of avidin conjugates is not uncommon. Streptavidin obtained fromStreptomyces avidin cultures has properties similar to those of avidin and binds biotin withvery high affinity. Streptavidin has a neutral pH and is considered superior to avidin withrespect to detecting biotinylated ligands.

Digoxigenin is a steroid isolated from digitalis plants such as Digitalis purpurea. It isan alternative to biotin for labeling hybridizing probes. Since digoxigenin is present only inthe blossoms and leaves of these plants, binding of the antidigoxigenin antibody does notoccur in any other biological specimens. It is thought that the efficiency of probe labelingis better with digoxigenin than with biotin, whatever technique is used (nick translation orrandom priming). This superiority is indicated by the observation that as little as l0pg ofdigoxigeninated albumin can be visualized in Western blot, whereas the limit of visualiza-tion for the biotinylated product is 500pg (Brunet et al., 1994).

The avidin-biotin system was developed for detecting antigens at the electron micro-scope level (Heitzmann and Richards, 1974). Later Heggeness and Ash (1977) proposedthe use of this system for fluorescence immunohistochemistry. Guesdon et al. (1979) pro-posed a variety of labeled avidin-biotin methods which were further supported by Warnkeand Levy (1980). The avidin-biotin methods used today are similar to the system describedby Hsu et al. (1981). This system is a significant improvement over the previous immuno-histochemical techniques. The problem of endogenous biotin is discussed on page 98.

Presently, nonradioactive probes, especially biotin or digoxigenin, are favoredbecause they are less hazardous to work with, can be more rapidly developed, and providebetter spatial resolution. Thus, introduction of nonradioactive detection systems has madeISH, using formalin-fixed and paraffin-embedded tissues, more accessible for applicationto molecular cell biology and diagnostic pathology. However, radioactive detection sys-tems are more sensitive than nonradioactive probes, especially oligonucleotide probesused instead of cRNA probes (Sperry et al., 1996).

The controversy over the degree to which radioactive probes are more sensitive hasnot been fully resolved. In any case, microwave pretreatment enhances ISH signal detec-tion of RNA and DNA whether radiolabeled or nonradioactive probes are used; both meth-ods are presented later. Furthermore, a number of approaches is available to increase thesensitivity of the nonradioactive ISH procedures (for a review, see Komminoth andWerner, 1997); some of these approaches are discussed below.

Compared with radioactive ISH, nonradioactive ISH requires a 10- to 50-fold higherconcentration of probes such as oligonucleotides. However, signal amplification isdecreased by increasing probe concentration. Therefore, since nonradioactive probes havelimited sensitivity, especially when applied to low-abundance mRNAs, a technique isrequired for signal amplification. One such technique consists of an optimized protocol forrapid signal amplification based on catalyzed reporter deposition (CARD) that increasesthe sensitivity of nonradioactive mRNA ISH on the formaldehyde-fixed and paraffin-embedded tissues (Speel et al., 1998). This technique facilitates the detection of low-copymRNAs by ISH (Yang et al., 1999).

The CARD approach uses horseradish peroxidase to catalyze the deposition of tyramidemolecules that are conjugated to an antihapten immunoglobulin or Streptavidin, which inturn, during the first incubation step, binds to the digoxigenin- or biotin-labeled nucleic acidtarget. Thus, the number of target sites for the next reaction step is markedly increased.

Other Applications of Microwave Heating 217

During this step, biotin sites on bound tyramine act as additional binding sites for antibi-otin enzyme. An additional round of signal amplification can be achieved by usingbiotinyltyramide and streptavidin conjugated to alkaline phosphatase (Yang et al., 1999).

Essentially, the CARD protocol is based on the deposition of haptenized tyramidemolecules in the vicinity of hybridized probes catalyzed by horseradish peroxidase. Thesuccess of this technique depends on the integrity of target mRNA in sections and the abil-ity of the probe to penetrate the sections and hybridize with mRNAs. Another requirementis an efficient reporter system capable of revealing low numbers of probe-mRNA hybridsper cell accompanied by low background staining.

Another approach to improve the resolution and detection sensitivity of the hybridiza-tion involves the use of semithin resin sections instead of paraffin sections

or frozen sections. The former shows better preservation of cytological detailsand higher spatial resolution. However, resin sections inhibit probe penetration into thesection, limiting probe hybridization to the target sequences/antibodies protruding fromthe section surface. Such sensitivity and probe penetration difficulties can be circumventedby using the methacrylate embedding–acetone deembedding (MEADE) technique (Warrenet al., 1998). These authors successfully localized mRNA and rRNA transcripts in marinebivalves. Other resins such as LR White and Lowicryl K4M do not allow tissue deembed-ding. Since this method requires longer incubation durations in acetone (12–15min) andalso a brief proteolytic digestion of the tissue to optimize intensity of the hybridizationsignal, such treatments tend to have an adverse effect on cell morphology.

Enhancement of in Situ Hybridization Signal with Microwave Heating(Sibony et al., 1995)

1.

2.3.

4.5.

6.7.

8.9.

10.11.12.

Fix human tissues with 4% paraformaldehyde in PBS for 24 hr, embed in paraf-fin, and cut 4- to sections.Mount sections on silane-treated glass slides.Deparaffinize with three changes of 5min each in xylene, followed by threechanges of 100% ethanol, two changes of 95% ethanol, and two changes of 75%ethanol.Rinse in 0.85% NaCl.Place slides in microwave transparent jars containing 0.01 M sodium citratebuffer (pH 6.0), and heat to the boiling point at maximum power (700 W) in amicrowave oven equipped with a rotating plate; cover the jars with loosely fittinglids unless 2–3 cm empty space is present above the buffer level in the jar.Allow boiling to continue for 7 min.Check the buffer level, add fresh distilled water if necessary, and again boilfor 5 min.Cool the slides to room temperature for 20 min.Rinse for 5 min in PBS (0.145M NaCl and 0.01M sodium phosphate buffer;pH 7.3).Postfix with 4% paraformaldehyde for 20 min.Rinse twice for 5 min each in PBS.Digest with proteinase K for 20 min.

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13.14.15.16.

17.

18.19.

20.

21.

22.

23.24.25.

26.

27.

28.29.

Rinse in PBS for 5 min.Again postfix with paraformaldehyde for 5 min and rinse in PBS for 5 min.Rinse in 0.85% NaCl.Dehydrate with increasing concentrations of ethanol in 0.3 M ammonium acetate,and air-dry.Hybridize by placing of the following solution for 16hr at 50°C.

This is done by lowering the sections in the hybridization mixture with a piece ofParafilm50% formamide10% dextran sulfate1mg/ml salmon sperm DNA 2 × SSC (1 × SSC contains 0.15 mol/liter sodiumchloride and 0.015 mol/liter sodium citrate)70mM DDTSense- or antisense-radiolabeled riboprobe (concentration adjusted to

to cpm/section)Remove Parafilms in 5 × SSC containing 10 mol/liter DTT.Wash with two changes of 30 min each in SSC (five times strength) at room tem-perature and 50°C, successively.Wash in SSC (double strength) containing 50% formamide and 10 mol/liter DTTfor 30min at 55°C.Treat with two changes of 10 min each in NaCl TE (0.5 M NaCl, 10 mM Tris-HCl[pH 7.5], and 5 mM EDTA).To reduce the background, remove all single-strand RNA molecules by digestionwith RNAse in NaCl for 20 min at 37°C.Treat with NaCl TE for 90 min at 37°C.Treat with 0.1 × SSC for 15 min at room temperature.Dehydrate with increasing concentrations of ethanol in 0.3 M ammonium acetate,and air-dry.Estimate and quantify macroscopically the hybridization signal on BIOMAX(Kodak, Rochester, NY) films after 5 days of exposure.After immersing the slides in liquid photographic emulsion NTB2 (Kodak),expose them in the dark for 2–6 weeks.Develop and fix photographically.Counterstain with toluidine blue.

Procedure for in Situ Hybridization of DNA

Paraffin sections of formalin-fixed tissues are mounted onto silanizedglass slides and air-dried at 60°C (Henke and Ayhan, 1994). They are deparaffinized, rehy-drated, and air-dried. The slides are placed in a plastic Coplin jar filled with l0mM sodiumcitrate buffer (pH 6.0) and microwaved (720 W) for 1 min; this time is measured after reach-ing the boiling point. After treating with 1 M sodium thiocyanate for 10 min at 80°C, thesections are washed with distilled water and then digested with pepsin (3mg/ml in 0.2 NHC1) for 6 min at 37°C. Both the heating and the pepsin digestion durations must be

Other Applications of Microwave Heating 219

adjusted to the respective tissue. Following washing twice for 5 min each in distilled water,the slides are air-dried and treated on a hot plate for 30 min at 80°C.

Each section is covered with of freshly prepared hybridization solution:Deionized formamide (65%)2 × SSC (0.3 M NaCl and 0.03 M sodium citrate)Dextran (10%)Salmon sperm DNABiotinylated probe DNASections are covered with cover-slips, sealed with rubber cement, denatured by heat-

ing at 78°C in a water bath for 10 min, and hybridized overnight at 37°C. The coverslipsare carefully removed by floating the slides in 2 × SSC, and the sections are washed twicefor 10 min each in a mixture of 2 × SSC and 50% formamide at 40°C, and then three timesin PBS. Endogenous peroxidase is blocked by incubation in 1% for 15 min.

The labeled DNA is detected by incubating the slides in PBS (10.4mM3.16mM 150mM NaCl, pH 7.6) containing 1.5% normal horse serum for 10 minat 37°C. The fluid is decanted, and a monoclonal mouse antibiotin antibody, diluted 1:100 inPBS, is added for 30 min at 37°C. The sections are washed three times in PBS and then incu-bated for 15 min at 37°C in a biotinylated goat antimouse antibody (Vector, Burlingame,CA), diluted 1:1000 in PBS containing 1% BSA. After three washes in PBS, the sections arecovered with peroxidase-conjugated streptavidin ( in PBS) for 30 min at 37°C. Thesections are carefully washed twice in PBS, and DAB (0.5 mg/ml in 0.05 M Tris-HCl buffer,pH 7.6, and 0.03% ) is added as the chromogen. The sections are counterstained withhematoxylin, dehydrated, and mounted. The results of this method are shown in Figure 9.4.

In Situ Hybridization of RNA in Skeletal Tissues

Skeletal tissues are mineralized and so require decalcification to obtain clear mor-phological details. Decalcifying reagents such as EDTA and HC1 have been traditionallyused at room temperature for processing such tissues for in situ hybridization. However,these and other similar agents require long durations of treatment, resulting in damagedcell morphology and reduced hybridization signals. It has been demonstrated that longdecalcification, for example with EDTA at room temperature, reduces hybridization sig-nals, while the signals are preserved better after treatment with the same reagent at thesame temperature for short-term decalcification (Kaneko et al., 1998).

The above-mentioned limitations can be significantly minimized by using microwaveheating. It has been shown that decalcification with EDTA in a microwave oven reducesthe duration for decalcification, which in turn prevents the reduction of hybridization sig-nals caused by long-term decalcification (Kaneko et al., 1998). Formic acid has also beenused in conjunction with microwave heating as a decalcifying agent and has been reportedto decalcify faster than EDTA (Callis and Sterchi, 1998). This treatment has not beentested for in situ hybridization. For in situ hybridization, each consecutive microwavedecalcification in the presence of 20% EDTA for 2hr at 50°C for 3–4 days is recom-mended. The rest of the time, the tissues are decalcified in 20% EDTA at room tempera-ture. A temperature lower than 55°C would safely preserve cell morphology.

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Other Applications of Microwave Heating 221

Microwave Heating for in Situ Hybridization of mRNA in Plant Tissues

Traditionally, processing of plant tissues for light microscopy required about 1 week.Although procedures are now available that allow paraffin embedding in short durations(Ruzin, 1999), anatomical preservation is less than excellent. To circumvent these prob-lems, Schichnes et al. (1999) introduced an efficient protocol using microwave heating forparaffin embedding and DIG-labeled (digoxigenin-UTP) knotted (kn) mRNA as a probe forhybridizing mRNA from the knotted gene in the meristematic tissues. This approachdecreases the durations of fixation from 24 hr to 15 min, dehydration from 73 hr to l0min,and infiltration from 96 hr to 3 hr. This procedure also minimizes the time required for sec-tion adhesion to slides as well as completion of staining. Moreover, anatomical preservationis superior, and localization of the mRNA probe is precise. This protocol is detailed below.

Fixation, Dehydration, and EmbeddingStep

1.

2.3.4.5.6.7.8.9.

10.11.

Fixation in PFA (4% paraformaldehydein PBS), repeat twice (cool on ice inbetween cycles for 1min)

NaCl (0.85%)Ethanol (50%)Ethanol (70%)Ethanol (70% + 0.05% safranin O)Ethanol (100%) repeat onceEthanol (50%) + isopropanol (50%)Isopropanol (100%)Isopropanol (50%) + molten paraffin (50%)Molten paraffinMolten paraffin, repeat four times

The total duration is 4hr.

Temperature inMicrowave Oven (°C)

3767676767677777776767

Time (min)

151.251.251.251.251.251.51.5

101030

Microwave Treatment

Areas of high microwave flux are checked with a Pelco 36140 microwave bulb array(Ted Pella). Specimens are not placed in areas indicated by illuminated bulbs. Vials con-taining the specimens are placed in a cold tap water bath (50 ml) that is preheated to therequired temperature. The temperature is regulated by placing the microwave temperatureprobe into a vial of the same solution that is present in the specimen vial. The built-in tem-perature probe displays the specimen temperature on the oven front panel. The wire thatattaches the probe to the oven is submerged in the water to decrease the antenna effect.An additional 400 ml of static water load is placed in the oven at an optimal position deter-mined with the microwave bulb array. This water is changed between every step.

Staining

Tissue sections ( thick) are placed on Probe-on Plus slides and floated on auto-claved water on a hot plate (42°C) for 2–3 min to remove compression. The water isremoved with a paper towel, and the slides are placed on a slotted glass staining dish onits side in the microwave oven. To adhere the sections, the slides are heated in a microwave

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oven at 43°C for 30 min. The probe is placed in a drop of water on a slide on top of theslide rack to measure the temperature.

The slides are placed in the staining dish which is filled with 1% safranin O, in 1 partmethyl cellusolve, 1 part 50% ethanol, 1% (w/v) sodium acetate, and 2% formalin solu-tion, placed in a water bath in the microwave oven, and loosely covered with plastic wrapto prevent splattering; tight-fitting covers must not be used. The temperature probe isinserted into the staining dish through the wrap. The slides are stained for 45 min at 60°Cin the oven.

Microwave Heat–Assisted Fluorescence in Situ Hybridization

If distinct hybridization signals from formalin-fixed paraffin-embedded tissue sectionsusing fluorescence in situ hybridization (FISH) are not obtained, the signals can beenhanced with an appropriate heat treatment. Such a treatment enables the FISH analysisof paraffin sections with poor or uncontrollable fixation conditions (including those ofarchival specimens) or other problems. Intermittent microwave heating of short durationscan rectify some of the problems encountered during conventional FISH. The conventionalFISH technique takes a long time to complete, whereas the new method in conjunctionwith intermittent heating can be completed in only 1 hr. Intermittent heating keeps the tem-perature as uniform as possible and prevents the sample from drying. This approach yieldsconsistent and distinct signals, without fluctuation in intensities and with minimal back-ground noise. In contrast, conventional FISH tends to result in background autofluores-cence that masks weak hybridization signals in the nuclei. Two applications of intermittentheating are given below.

Intermittent microwave heating has been used for enhancing FISH signals in theparaffin-embedded tissue sections of gastrointestinal neoplasia (see page 458) (Kitayamaet al., 2000). The processing of these tumors is known to be particularly difficult becauseof the presence of necrosis and contamination with inflammatory and normal stroma cellsand poor attachment of paraffin sections to the slide.

The second example of the use of the intermittent heating is for identifying cen-tromeres in gastric cancer cells (Kitayama et al., 1999). A panel of 17 centromeric specific

probes was used for detecting chromosomal instability in these cells. The studyof centromeres is important because chromosomal abnormalities are a well-known char-acteristic of human cancers.

Procedure for Gastrointestinal Neoplasia

Gastric tumor tissue is fixed with 4% neutral formaldehyde for 1 day and embedded inparaffin (Kitayama et al., 2000). Paraffin sections ( thick) are deparaffinized with xyleneand rehydrated with ethanol. Centromeric DNA probes and locus-specific identi-fier probes (c-myc and p53) are available from Vyis Inc. (Downers Grove, IL). The probesare labeled with orange (Cy 3) or green (FITC) using digoxigenin-11-dUTP and nick trans-lation. The sections are placed in 0.01 M citrate buffer (pH 6.0) and heated in a microwaveoven for l0 min. This is followed by treatment with 0.2% pepsin in 0.01N HC1 for l0 min at37°C, and then exposure to 0.1% NP-40/2 × SSC for l0 min at the same temperature.

Other Applications of Microwave Heating 223

The DNA in the sections is denatured by treatment with 70% formamide/2 × SCC for5 min at 80°C. Ten microliters of the probe solution (hybridization buffer: probe:and distilled water: ) is placed on the slide and coverslipped. The slide is placed in amicrowave oven (2.45 GHz, 300 W) and heated for 3 sec at 2-sec intervals for a total of15 min at 42°C. DAPI II (4,6-diamidine-2-phenylindol) (125 ng/ml) is used for nuclearstaining. The sections are promptly observed under a fluorescent microscope equippedwith epifluorescence filters and a photometric CCD camera. The captured images aredigitized and stored in an image analysis program.

The number of signals per cell is counted for a total of 100–200 cell nuclei, and thesignal intensities of the different periods of hybridization are simultaneously compared.

Nuclear Fluorescence in Situ Hybridization Signal UsingMicrowave Heating

To avoid the limitations of enzyme digestion, microwave heating alone can be usedfor in situ hybridization. Microwave boiling facilitates the probe’s penetration through thetissue to the nucleus. Also, this treatment causes at least partial denaturation of the nuclei,enhancing hybridization. Microwave heating alone has been employed for fluorescencein situ hybridization for chromosomal sequences (Bull and Harnden, 1999). The pretreat-ment time is reduced to ~ 1 hr. Prostate tissue is fixed overnight with 4.4% formaldehydecontaining 1 % NaCl (pH 6.3). Paraffin sections ( thick) are placed on slides which canbe stored at room temperature.

The sections are deparaffinized, followed by hybridization. The slides are placed ina glass staining jar containing a few antibumping granules (BDH Lab Supplies, Poole,England), filled to the brim with a mixture of 100 mM Tris-HCl buffer and 50 mM EDTA(pH 7.0), and placed at the center of a rotary 800 W microwave oven. Microwave boilingis carried out at full power for 2–3 min. For four cycles of microwave heating, hot fluid(65°C) is used to refill the staining jar as rapidly as possible to avoid drying of the slides.The slides are transferred to 70% ethanol at 4°C and then to 100% ethanol at the sametemperature, followed by air-drying.

Digoxigenin-labeled chromosome 10 probe (DIOZI; Oncor, Gaithersburg,MD) is used at a final concentration of in hybridization buffer (50% formamide,10% dextran sulfate, 0.004% Tween 20, and standard saline citrate (SSC) [in 1.5 strength]).A volume of the probe is placed on an 18 × 18-mm coverslip, which is placed ontothe slide, sealed with special Vulcanizing Fluid, and placed on a flat-bed thermal cycler.Slides are incubated for 3 min at 94°C and placed in a humidified box for 16 hr at 37°C.

Coverslips are removed, and slides are placed in wash buffer (4 × SSC/0.05% TritonX-100) twice for 2 min each. This is followed by a thorough wash in 0.25 × SSC for 5 minat 72°C, transfer to wash buffer at room temperature, and flooding with blocking buffer(0.5% milk powder in wash buffer) for 5 min. The signal is detected for 30 min at 37°Cwith antidigoxigenin, diluted 1:100 in blocking buffer. Coverslips are gently removed,and slides are rinsed three times for 2 min each in wash buffer at 45°C before mountingin antifade (Vectashield, Vector Lab., Burlingame, CA) containing propidium iodide

Slides are viewed on an Axioskop microscope (Carl Zeiss) equipped with astandard camera and appropriate software (PSI, League City, TX).

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MICROWAVE HEAT–ASSISTED POLYMERASE CHAIN REACTION

The polymerase chain reaction (PCR) technology yields a DNA fragment for cloningand is especially useful for cloning cDNAs. This technique uses the enzyme DNA poly-merase to make a copy of a defined region of DNA. The region of the DNA we want toamplify is selected by putting in short pieces of DNA primers that hybridize to DNAsequences on either side of the selected region and cause initiation (priming) of DNA syn-thesis through that region. The copies of both strands of the selected region, as well as theoriginal DNA strands, serve as templates for the next round of amplification. Thus, theamount of the selected DNA region doubles again and again with each cycle.

The PCR technique can be used to study DNA from a variety of sources, includingarchival specimens containing formalin-fixed, paraffin-embedded tissues (Alcock et al.,1999). Microdissection can be performed on sections cut from such tissues that have beenprocessed for conventional immunohistochemistry. Crude DNA extracts, obtained frommicrodissected specimens by microwave heating, can be added directly to amplificationreactions. Analyses using a range of PCR-based techniques, including microsatellite repeatpolymorphism analysis at the NM23-H1 locus and sequencing of exon 5, 7, and 8 of thep53 gene, can be performed.

Procedure

Sections of formalin-fixed, paraffin-embedded tissues mounted on slides aredeparaffinized with two washes of xylene for 5 min each and rehydrated in a graded seriesof ethanol. Endogenous peroxidase activity is quenched by treating the sections with a0.3% solution of in methanol for 20 min. Standard immunohistochemistry is per-formed with a Vector stain Elite kit (Vector Laboratories, Burlinghame, CA), using pri-mary antibodies against the protein products of the p53 oncogene (DO7; Novocastra, NewCastle upon Tyne, UK) and the putative metastasis suppressor NM23-H1 (anti-nm23-Hl/NDPK-A; Novocastra). Immunoreactivity is visualized with DAB, followed by coun-terstaining with hematoxylin. For immunohistochemistry of p53 antigen, the sections areplaced in 0.01 M trisodium citrate fluid for 8 min at 800 W and then allowed to cool to50°C. The temperature during antigen retrieval is not allowed to exceed 85°C.

Following immunohistochemistry, the sections are kept covered with deionized waterin a humidified chamber until microdissection on a Leitz Laborlux microscope at X100magnification. Excess water is drained from the slide, and the area surrounding the sectionis dried carefully. About of 1 × TE buffer (10 mM Tris, 1 mM EDTA) is placed overthe section to create a bubble of liquid over the section. Areas of interest are microdis-sected with a disposable microlance 3 needle attached to a 10-ml syringe, and harvestedwith a pipette. Using multiple samples from a section require thorough washing withdeionized water to prevent cross-contamination of microdissected samples.

The microdissected section fragments in the TE buffer are transferred from the slideto a 0.5-ml plastic tube and centrifuged at 12,000 g for 15 min. The supernatant is dis-carded, and the tissue pellet is resuspended in of mineral oil and heated in amicrowave oven at 800 W for 7 min. After resuspension in an aliquot of TE buffer, the sam-ple is added to the master mix for PCR amplification. The details of amplification andsequence analysis are presented by Alcock et al. (1999).

Other Applications of Microwave Heating 225

DETECTION OF ANTIGENS BY FLOW CYTOMETRY

Flow cytometry has been a versatile method for detecting and quantifying cell surfaceantigens in diagnostic and research fields for many years. Recently, it has also become use-ful for simultaneous detection of cytoplasmic and nuclear antigens by using optimal fixa-tion and cell permeabilization protocols. Generally, cell fixation and cell membranepermeabilization are mandatory for the detection of intracellular antigens. Fixation can becarried out with an alcohol or an aldehyde, and cell membrane permeabilization can beaccomplished by treating the cells with detergents such as Tween 20, Triton X-100, saponin,lysolecithin, or NP-40. Detergents facilitate access of large molecules such as antibodiesand DNA fluorochromes to the target antigen after or before the cells have been fixed.

Care is required to achieve optimal cell membrane permeabilization. If permeabiliza-tion is insufficient, the accessibility of antibodies to the target antigen will be hampered,resulting in underscoring (Lan et al., 1996). If permeabilization is excessive, cell mor-phology will be damaged, causing a nondiscrimination pattern in cell populations.Consequently, it is difficult to use light scatter signals to gate on the cells for accurateanalysis. Since optimal fixation and detergent permeabilization depend on the type of celland the characteristics of the antigen under study, these parameters are determined by trialand error. Thus, this approach is somewhat tedious and time consuming.

Image cytometric quantitation of nuclear immunostaining can be carried out with theCAS 200 image cytometer (Becton Dickinson, San Jose, CA). It is based on differentialstaining of nuclei for the antigen (e.g., Ki-67 in ductal carcinoma of the breast usingmicrowave heating and MIB-1 antibody) (Bhoola et al., 1999). Immunopositive nuclei arebrown, while negative nuclei counterstain as blue with hematoxylin. Using the two CAS200 sensors, measurements are made at different wave lengths. At 620 nm, both brown andblue absorb, providing a mask of all nuclear material. At 500 nm only brown stain absorbs,allowing the positive nuclei to be measured independently. Comparison with the 620-nmmask gives a percentage of nuclei area stained positively.

The image cytometer is standardized by adjusting the light source of the microscopeto a predetermined value on an empty field. Then, in control mode and on the negative con-trol slide, the antibody threshold is adjusted using nuclear areas considered negative. Aftercomparing the brown mask with the blue and the brown-stained image seen through themicroscope, the slide stained for the antigen is analyzed in the specimen mode.

Fifteen high-power fields are analyzed in each case, using random but consecutivefields when possible. Tumor cells are isolated from stroma by using the scene segmenta-tion function, which allows the operator to precisely define portions of the image to be ana-lyzed (Bhoola et al., 1999). Computer-generated histograms show the percentage ofpositive nuclear area on the vertical axis and nuclear optical density on the horizontal axis.

Microwave Heat–Assisted Flow Cytometry

The limitations of the application of conventional detergents mentioned above can becircumvented by replacing this approach with cell membrane permeabilization bymicrowave heating. Improved detection of intracellular antigens can be obtained withmicrowave heating used in combination with flow cytometry. This approach yields his-togram patterns that show clear discrimination between intact cells and cell debris (Fig. 9.5).

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Accurate flow cytometric measurements depend on clear discrimination between intactcells and cellular debris. Both cytoplasmic (bcl-2, CD68, lipocortin-1, muscleactin, and desmin) and nuclear antigens (p53, PCNA, Ki-67) have been simultaneouslydetected with this combined protocol (Lan et al., 1996; Millard et al., 1998).

It has been mentioned earlier in this volume that microwave heating facilitates anti-body access to target antigens by breaking down protein crosslinks introduced byformaldehyde during fixation. Microwave heating also preserves cell morphology satis-factorily. Other advantages of this method are easy reproducibility and simultaneousdetectability of a number of intracellular antigens. Thus, it has more general applicationthan that of other methods. Although conventional detergent permeabilization can retrievecertain antigens (Teague and El-Naggar, 1994), some other antigens are not detectable. Itmeans that this approach is limited in its application in that each of the detergents allowsdetection of a limited type of intracellular antigen.

According to Millard et al. (1998), improved detection of intracellular as well as sur-face antigens can be accomplished with flow cytometry using two commercially availablechemical reagents, the ORTHOPermeaFix (OPF) and FIX&PERM Cell PermeabilizationKit (F&P). OPF is an aldehyde proprietary mixture of reagents that is commercially avail-able (ORTHO Diagnostic Systems, Raritan, NJ). After fixation of cells with OPF,immunostaining and flow cytometric analysis can be delayed, if necessary, for at least1 week. Gentle fixation with this reagent can be accomplished in 45 min to 24 hr withoutany adverse effects (Pizzolo et al., 1994). This property has a practical advantage in thatsmaller laboratories can collect a patient’s samples to be subsequently sent to a well-equipped laboratory for investigation. Such arrangements also facilitate internationalcollaboration.

Other Applications of Microwave Heating 227

It is thought that the microwave heating method is comparable to the OPF and F&P pro-tocols, though the former method is inexpensive, which is important because in a diagnosticlaboratory cost is a consideration in choosing a particular method. Moreover, the exact ingre-dients of OPF and F&P are not known. It should be noted, however, that unlike these com-mercial reagents, microwave heating may damage or cleave sensitive cell surface antigens,preventing cell selection on the basis of combined cell membrane and intracellular antigens,but microwaving decreases nonspecific background fluorescence (Millard et al., 1998).

Procedure 1

Cells are washed in PBS and fixed with 2% paraformaldehyde for 30 min at4°C. The supernatant is discarded after centrifugation (500 g for 5 min at room tempera-ture), and cells are resuspended in 10 ml of 0.01 M sodium citrate buffer (pH 6.0) contain-ing 0.5% bovine serum albumin in an unsealed 50-ml propylene tube. The tube is placedupright in the center of a 1-liter Pyrex glass beaker, which is sealed with polyethylene plas-tic wrapping. The cell suspension is heated for 30–60 sec in a microwave oven at the max-imum power setting (800 W), reaching a temperature between 90 and 100°C. The cells arechilled on ice for 10 min. After centrifugation at 500 g for 5 min, the supernatant is dis-carded and the cells are washed in PBS. The cells can be filtered through a meshto remove the aggregated debris and then labeled with monoclonal antibodies for flowcytometry as described below.

The cells are incubated in the primary antibody at an appropriate dilution for30–60min at 4°C or room temperature, depending on the type of cells or antigens. Afterbeing washed in PBS, the cells are incubated in a fluorescein isothiocyanate (FITC)–con-jugated goat antimouse IgG (or sheep antimouse IgG) for ~30 min at 4°C in the dark. Thecells are washed twice in PBS and resuspended in of 1% fetal calf serum orISOTON II for flow cytometric analysis.

The cells are run on a flow cytometer, an EPICS 752 (Coulter Electronics, FL) con-nected to a CICERO data acquisition system (Cytomation, CO) or FACScan (BectinDickinson). An argon ion laser (Coherent, CA) operating at 488 nm is used to illuminatethe cells. Forward and right-angle light scatter signals are collected along with FITC fluo-rescence (measured through a 535-nm bandpass and logarthimic amplification) and PI flu-orescence (630 nm long-pass filter) where appropriate. Fluorescence histograms of at least5,000 counts are generated from a gate set in the forward angle versus 90°C light scatterscattergram. The percentage of positive cells is measured from a cutoff set using anisotype-matched, nonspecific control antibody, while the mean channel fluorescence ismeasured over the entire distribution. Figure 9.5 shows clear discrimination between intactcells and cell debris after microwave heating.

Although the above method is highly recommended, if the expression of immuno-staining is weak because of antigen masking and inaccessibility of antigens to antibodiesin the aldehyde-fixed and paraffin-embedded tissues, single cell/nuclei can be isolatedfrom archival paraffin-embedded tumors for laser flow cytometry using fluorescent-labeled primary or secondary antibodies. This approach is especially useful for steroid hor-mones such as estrogen and progesterone, which have nuclear binding sites. The advantageof nuclear isolation is the greater accessibility of immunoreagents to the nuclear proteinscompared with that in the nuclei of whole cells.

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Another advantage is the recognition of two subpopulations with low and high stain-ing. Such a heterogeneity of the nuclear antigenic expression (e.g., estrogen) is not seen inthe whole cell preparation analyzed by flow cytometry. This heterogeneity is possibly dueto improved reactivity and maximum access of the nuclear proteins to the antibodies.Heterogeneity of nuclear protein expression is thought to be due to the variations in thecontent of nuclear proteins, their cell cycle stage, and proliferation (Sabe et al., 1999). Inthis respect, different physiological states and differences in size and surface charge of theprotein are also important.

The nuclear isolation method has been used for processing archival paraffin-embeddedmammary tumors for monitoring estrogen expression and aneuploidy (Sabe et al., 1999).These two parameters have important diagnostic and prognostic significance in mammarytumors.

Procedure 2

Approximately sections cut from formalin-fixed and paraffin-embedded tis-sues (e.g., breast) are treated with 0.05% pepsin (cat. no. P7012, Sigma) in normal saline (pH1.65) for 1 hr at 40°C (Sabe et al., 1999). The sections are vortexed every 5 min for an additional30 min. This proteolytic reaction is terminated by adding 5 ml of cold 10% fetal bovine serum(FBS) in ethylene-diaminetetraacetic acid (EDTA). After filtration through nylon mesh,the filtrate is forced through a syringe with a 28-gauge needle and centrifuged at 300g for7 min. The resulting pellet is resuspended in 1ml of nuclear isolation medium (Hank's PBSwith 0.2% FBS, 25 mM HEPES buffer, and 0.6% NP-40) for l0 min at 4°C.

The cells and nuclei are aliquoted into polystyrene tubes. Approxi-mately of normal horse serum (Vector Laboratories, Burlingame, CA) is added toblock any nonspecific binding. The suspension is incubated with biotinylated antiestrogenmonoclonal antibody (1D5, Dako Corp., Carpinteria, CA) at 1:25 dilution for 1 hr at 37°C.Aliquots are stained with of fluorescein isothiocyanate (FITC)–conjugated strepta-vidin (Dako buffer containing 0.1% Triton X-100).

The suspensions are centrifuged and washed twice in 3% FBS/PBS. They are againcentrifuged and washed twice in 3% FBS in PBS with 0.1% Triton X-100. The pellets areresuspended and incubated with 0.5 ml of 1% FBS containing propidium iodide (Calbiochem, San Diego, CA) and RNAse (1 mg/ml) in Hank’s balanced salt solution (with-out phenol red) for 30 min at 37°C. The samples are analyzed on a Coulter ElectronicsXL-MCL or a Becton Dickinson FACScan flow cytometer with standard argon ion laserexcitation and filter configuration for the FITC/propidium iodide dye combination.

MICROWAVE HEAT–ASSISTED ENZYME-LINKEDIMMUNOSORBENT ASSAY

Microwave heat–assisted enzyme-linked immunosorbent assay (ELISA) has beenused for measuring anti-glomerular basement membrane (GBM) antibodies in kidneyserum (Van Dorp et al., 1991) The presence of GBM antibodies is one of the characteris-tics of Goodpasture’s syndrome. The application of microwave heating reduces the durationof incubation to assay circulating anti-GBM autoantibodies.

Other Applications of Microwave Heating 229

Antigens in human kidney serum are diluted 1:500 in a solution (0.1M0.1M and 0.02% Na azide, pH 9.6), and of this dilution is placed in eachwell of the 96-well microtiter plate. The incubation is carried out for 2 hr at 37°C, and dur-ing this period the dilution is exposed to microwave heating for 15 min at a power level of~ 290W (37.1°C) in the Bio-Rad oven. The power level will differ depending upon themicrowave oven used and the position of the microtiter plate in the oven. The plate is posi-tioned on a 5-cm-thick polystyrene platform in the center of the oven to absorb excessmicrowave energy.

The serum is diluted 1:25 in PBS (pH 7.2) containing 0.5% Tween-20 and 1%

conjugate is diluted appropriately in PBS containing 0.5% Tween 20 and 1% and100 ml of the conjugate is placed in each well. It is exposed to microwave heating for 15 minas above.

Conventional incubation is carried out by placing 100 ml of the substrate (0.02%0.04 mg/ml OPD, 0.04M citric acid, and 0.05 M disodium hydrophosphate, pH 5.0) in eachwell for 20 min at room temperature. The reaction is stopped with of 4 N-sulfuricacid per well. The optical density of the colored solutions is read at 492 nm with amultiphotospectrometer.

MICROWAVE HEAT–ASSISTED SCANNING ELECTRON MICROSCOPY

Like other specimens, bacteria can be processed for scanning electron microscopy(SEM) using microwave heating. Conventional processing of specimens for SEM is car-ried out in ~4 hr, while they can be prepared in ~1 hr using microwave heating (Fox andDemaree, 1999). Bacterial cells at an early exponential growth phase on polycarbonatemembrane filter (Nucleopore Corporation, Pleasanton, CA) can be used. The membranefilter with attached cells is cut into small squares (~50 × 50 mm) and transferred topolypropylene Petri dishes (60 × 15 mm), which are then placed in the cold spots in themicrowave oven at power level four (536 W). Previous to this step, using the neon bulbarray, cold spot determination has been done. Also, two beakers containing water havebeen placed in the microwave oven as water loads to absorb microwave energy. The fixa-tion is accomplished by placing ~ 1–2 ml of for 20 sec in the Petri dish.

The temperature probe is placed into a blank polypropylene Petri dish during pro-cessing, and the temperature is restricted to 37°C to prevent overheating. The filtrate isrinsed three times for 5 min each with 1–2 ml of phosphate buffer. The sample is dehy-drated in a microwave oven with 1–2 ml of an ethanol series of increasing concentrations(once in each of 50%, 70%, and 90% ethanol and three times in 100% ethanol). Each dehy-dration step is carried out for 20 sec in the microwave oven at power level 4 (536 W) witha temperature restriction of 37°C utilizing the temperature probe placed in the blank Petridish containing ethanol.

The filtrate is further dehydrated in 100% hexamethyldisilazine (HMDS) for 20 sec at37°C in a microwave oven at the same power level utilizing the temperature probe in theblank Petri dish containing HMDS. The filtrate is dried in a conventional oven for 15 min at60°C. After 15 min all excess HMDS is removed, and the filter with attached cells is allowed

and of the dilution is placed in each well. It is exposed to microwave heating for15 min at 41.1°C at the same power level as above. The monoclonal antibody HB43-HRP

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to continue to dry in the oven. The samples are sputter-coated with gold (1.5 min at 40 mA)and then viewed in a scanning electron microscope. The whole process takes ~ 1 hr. Theresults of this procedure, as shown in Figure 1.2, are similar to those obtained with stan-dard technique. For additional information on the microwave heat–assisted processing ofspecimens for scanning electron microscopy, the reader is referred to Demaree (2001).

MICROWAVE HEAT–ASSISTED CONFOCAL SCANNING MICROSCOPY

Confocal laser scanning microscopy can be used in conjunction with microwave heatingfor examining the three-dimensional structure and cellular interrelationships in sections ofparaffin-embedded tissues (Boon and Kok, 1994). Tissues are fixed with Kryofix, a coag-ulant fixative containing 50% ethyl alcohol and polyethylene glycol (PEG; molecularweight 300) for 90 sec in a microwave oven. The use of thick paraffin sections andfluorescently labeled antibodies is preferred.

MICROWAVE HEAT–ASSISTED CORRELATIVE MICROSCOPY

The choice of tissue processing method is crucial for optimal detection and quantifi-cation of target antigens in cells by immunogold light and electron microscopy. The primarycriteria for choosing a method are efficient, specific, and reproducible labeling of the antigenand satisfactory preservation of cell morphology. In some cases correlative light andelectron microscopy for analyzing immunostaining is desirable. These objectives can beachieved by observing semithin and thin sections of the tissue embedded in water-miscible(e.g., Lowicryl) or water-immiscible (epoxy resins) media. Semithin sections allow a sur-vey of the spatial distribution of the antigen, and thin sections provide subcellular expres-sion of the antigen on consecutive sections. Immunostaining of both semithin and thinepoxy sections can be enhanced by controlled etching of sections with sodium ethoxide(0.6% hydrogen peroxide in 96% ethanol) followed by antigen retrieval in a microwaveoven. This procedure was recently used for immunostaining of E-cadherin, and

in the human proximal jejunum (Groos et al., 2001).One of the advantages of using an epoxy resin is that, in contrast to cryosections, each

tissue block can be repeatedly sectioned for both light and electron microscopy. Treatmentwith ethoxide permeabilizes the section surface by partial corrosion of the embeddingresin. It is essential to determine the optimal concentration of the etching agent and etch-ing duration to obtain sufficient permeability of the section surface for antibody accesswhile avoiding structural damage. It is also necessary to find out optimal heating treatmentfor unmasking antigens hidden by covalent bonds formed between epoxy resin and bio-logical material during polymerization.

Procedure

Tissue specimens are fixed with 4% formaldehyde (freshly prepared fromparaformaldehyde) for 12–18 hr at 4°C, and embedded in Epon (Groos et al., 2001).

Other Applications of Microwave Heating 231

Semithin sections ( thick) are collected on silane-treated slides and air-dried atroom temperature. The controlled corrosion of the sections surfaces is obtained by treat-ing them with sodium ethoxide for at least 15 min, then rehydrating them with descendingconcentrations of ethanol. Antigen retrieval is achieved by transferring the sections in0.01 M citrate buffer (pH 6.0) and heating three times for 5 min each in a microwave ovenat 700 W, followed by cooling to room temperature for 30 min.

Nonspecific protein binding is blocked by treating the sections with 5% BSA dis-solved in PBS for 30 min at room temperature. After a brief rinse with PBS, the sectionsare incubated overnight at 4°C with the primary antibody diluted appropriately in PBS con-taining 1% BSA. They are rinsed in PBS, treated for 1 hr with biotinylated goat antimouseIgG and then treated with peroxidase-labeled streptavidin for 30 min.Peroxidase labeling is visualized with DAB as the chromogen. The sections are rinsed inPBS followed by in rinsing in distilled water, dehydrated in ascending concentrations ofethanol, cleared in xylene, and mounted.

Thin sections (~70 nm thick) from the same tissue block are collected on nickel grids.Incubation steps are carried out by floating the grids on drops of each solution, andwashing steps are performed by dipping them into the washing solution. Thin sections arecorroded by etching with saturated sodium ethoxide diluted to 50% with absolute ethanolfor 10 sec. After rehydration and washing in distilled water, antigen retrieval is carried outby heating the sections for 10 min to 95°C and then cooling them to 21 °C at a rate of0.04°C/sec in a thermocycler.

Following washing in three changes of PBS, nonspecific protein binding is blockedby treating the sections with 5% BSA in PBS for 30 min. Incubation with the primary anti-body is carried out overnight at 4°C. After application of the 10-nm gold-labeled second-ary antibody for 1 hr at a concentration of and subsequent washing in PBS, theimmunoreaction is stabilized by treating the sections with 2.5% glutaraldehyde in PBS for10 min. The sections are counterstained with uranyl acetate and lead citrate. Control sec-tions are processed the same way as the experimental sections, except that either the pri-mary antibody is omitted or is replaced with normal mouse serum in the same concentrationas the applied antibody.

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Chapter 10

Cell Proliferating Antigens

Some cell-proliferating antigens that are detected with antigen retrieval methods usingfrozen or formalin-fixed and paraffin-embedded tissues are discussed below. They includecell nuclear proliferating antigens (Ki-67 and proliferating cell nuclear antigen [PCNA]),p53, estrogen, androgen, and progesterone. Such immunohistochemical studies are importantfor diagnostic, prognostic, and therapeutic purposes. These studies are in common use toassess the importance of several antigens as prognostic factors in all kinds of malignan-cies. This approach is more commonly used in pathology laboratories than is analysis ofgene mutation at the molecular genetic level, which is cumbersome and time consuming.

Before discussing cell nuclear proliferating antigens, it is relevant to briefly explain thecell cycle. Proliferating cells can occupy several functional states besides mitosis. After com-pleting mitosis, the daughter cells enter the Gap 1 phase. The duration of phase varieswith the tissue type. Subsequently, cells enter the S (synthetic) phase, where the cell’s geneticmaterial is doubled during DNA synthesis. This phase is followed by a second Gapphase before cells divide again. The durations of these phases in descending order are(8–10 hr), S (6–8 hr), (4–6 hr), and mitosis (30–45 min). Proliferating cells after thephase leave the cell cycle, cease proliferation, differentiate, and eventually die or they entera resting phase from which they may be recruited back into the cell cycle at a later time.

KI-67 ANTIGEN

Ki-67 is a highly positively charged alkaline nonhistone protein (pI = 9.9) with repet-itive elements and a high content of randomly distributed prolines (8.2%) and lysines(11.4%) (Duchrow et al., 1994). It is encoded by a single gene on chromosome 10. Theprotein is a bimolecular complex of molecular weight 345 and 395 kDa. Ki-67 monoclonalantibody detects two polypeptides of these molecular weights in proliferating cells. SinceKi-67 protein contains many proline–glutamic acid–serine–threonine (PEST) motifs, thisprotein is capable of being very rapidly catabolized (Rogers et al., 1986). Thus, it has ahalf-life of only 1–2 hr. Cloning and sequencing of the complete cDNA of Ki-67 antigenhave been carried out (Duchrow et al., 1994).

The Ki-67 antigen was originally identified by its cell-cycle-related expression and isnow considered to be a more specific marker for cell proliferation and cell cycling than isPCNA (Gerdes et al., 1984). This is supported by the observation that ependymal cells,

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which are unable to regenerate, are PCNA-positive, but Ki-67-negative (Sarnat, 1995;Funato et al., 1996).

Ki-67 antigen is expressed throughout all phases of the cell cycle ( S, and M),except the quiescent phase. This means that the expression of this antigen is inti-mately associated with the cell cycle. The topographical distribution of Ki-67 antigen isalso cell cycle-dependent. Its expression becomes apparent at the beginning of the

phase and accumulates within the nucleus (predominantly in the perinucleolar region) dur-ing the late phase. The expression of this antigen increases as the cell cycle progresses,reaches its maximum concentration at mitosis, and markedly diminishes thereafter. In fact,immediately after mitosis its amount is minimal.

In the phase Ki-67 antigen is predominantly located in the nucleoli and is partic-ularly evident in cells containing relatively large nucleoli. Treatment with DNase or RNaseindicates that the antigen located in the nucleoli is associated with RNA (Szekeres et al.,1995; Benfares et al., 1996). In the later phases of the cell cycle it is also detected through-out the nucleoplasm, being found mostly in the nuclear matrix, where it is associated withDNA. During mitosis, it is present on all chromosomes and appears in a reticulate struc-ture surrounding the metaphase chromosomes (Verheijen et al., 1989). The chromosomalbinding of Ki-67 antigen is considered to be due to electrostatic attraction. Because of theshort half-life of Ki-67, it is rapidly degraded, resulting in decreased immunostainingduring anaphase and telophase.

Ki-67 antigen is also sensitive to the nutritional status of cells, declining rapidly after3 days of nutrient depletion in mitotic cells or becoming undetectable upon nutritional dep-rivation in lung cancer cells (Verheijen et al., 1989; Tinnemans et al., 1995). However,according to Dong et al. (1997), a greater relative change in Ki-67 expression occurs underconditions where proliferation is inhibited without growth fraction change than under con-ditions where a significant change in growth fraction does occur. It should be noted thatthese two observations were made using different cell types.

It is apparent from the above discussion that Ki-67 is present in the nucleus of prolif-erating cells and is an indicator of the growth fraction in tumor cells. It is primarily a DNA-binding protein that plays a crucial role in the maintenance or regulation of cell division.This protein may also function as a matrix for chromosomal DNA or contribute to the con-densation of the chromosomes or be involved in breakdown of the nuclear membrane beforemitosis (Duchrow et al., 1994). The association of Ki-67 with RNA in the nucleoli and withthe DNA with nuclear matrix suggests that the antigen plays a role in transcriptionalprocesses as a structural protein by mediating between nuclear DNA and nucleolar RNA.

Ki-67 antigen is a valuable tool for measuring cell growth in human tissues and cellcultures, particularly with respect to the histopathological determination of malignancy. Itcan provide information on the fraction of actively cycling cells. In certain malignanttumors the number of tumor cells immunohistochemically positive for Ki-67 antigen coin-cides with estimated tumor proliferation rates (Gerdes et al., 1983). In fact, this antigen isabsolutely required for maintaining active cell proliferation. In addition, immunohisto-chemical evidence indicates that Ki-67 antigen may be associated with neurofibrillarydegeneration in Alzheimer’s disease, other neurodegenerative disorders, normal agedbrains, and neoplasms such as gangliogliomas (Smith and Lippa, 1995). This antigen pos-sibly plays a role in the production of abnormally phosphorylated tau protein, which leadsto the formation of paired helical filaments within susceptible neurons. Considering these

Cell Proliferating Antigens 235

functions, Ki-67 antigen is expected to be expressed in all active parts of the cell cycle.Unlike PCNA, Ki-67 is not involved in DNA repair.

Ki-67 has a much shorter half-life (1–2 hr) than PCNA. Therefore, unlike the latter,the former demonstrates the proliferative stage of the cell rather than the residual evidenceof the cell that has passed through the cell cycle stage. It has been demonstrated that nor-mal tissues adjacent to carcinoma are PCNA-positive but Ki-67-negative (Wolf andDittrich, 1992). On the other hand, in an immunohistochemical study, Ki-67 expressionwas of no predictive value in squamous cell carcinoma of the head and neck (Roland et al.,1994). A recent study also indicates that the expression of Ki-67 in primary intraoral squa-mous cell carcinomas of the head and neck is independent of tumor site (Nylander et al.,1997). It has also been demonstrated that Ki-67 antigen staining might contribute to falsedata on the growth fraction (Ansari et al., 1993). If this problem arises, it can be resolvedby employing double labeling with two markers, Ki-67 and statin, for proliferating cellsand resting cells, respectively. However, overwhelming evidence has established Ki-67immunohistochemistry as an important tool for assessing cell proliferation in situ.It should be noted that, like most other antigens, the detectability of Ki-67 is highlyinfluenced by fixation and other preparatory parameters.

Immunohistochemistry

Ki-67 antigen immunohistochemical staining is a simple and reliable procedure forstudying tumor proliferative activity in frozen or formalin-fixed, paraffin-embedded tis-sues, including archival specimens. This antigen can be retrieved on sections of formalin-fixed and paraffin-embedded tissues by autoclave treatment (Fig. 10.1) or microwaveheating (Fig. 10.2). Both methods are reliable and are presented later.

Several antibodies against Ki-67 antigen have been generated (Gerdes et al., 1983,1984; Key et al., 1993; Kreipe et al., 1993), which are discussed later. The direct diagnosticand independent prognostic value of Ki-67 antigen staining using these antibodies has beenwell documented. Although Ki-67 antibody has been extensively used and is still beingemployed, MIB-1 antibody is preferred because it is equally effective in obtaining immuno-staining of Ki-67 antigen in frozen or fixed tissues. Fixed specimens show better preserva-tion of cell morphology, allowing clear distinction between positive and negative cells.

The above-mentioned advantage also facilitates quantitative immunostaining studiesof recently fixed tissues, as well as retrospective examination of archival specimens whichhave been stored after fixation and embedding. For example, immunostaining of archivalspecimens (1960–1992) using MIB-l antibody has been carried out for predicting the clin-ical outcome in patients with acinic cell carcinomas of salivary gland origin (Skalova et al.,1994). This antibody has also been used for immunostaining Ki-67 antigen inhuman proliferating bone tumor cells using autoclave treatment (Tsuji et al., 1997). Thismethod allows retrieval of this antigen in both formalin-fixed and ethanol-fixed specimens.

Note that although immunohistochemistry is contributing significantly to clinicalinformation, in the absence of quantitation, this method is subjective and prone to intra- andinterobserver variations. To assure the reproducibility of histopathological results and theircorrect evaluation, at least an assessment of the percentage of positive cells and both thestaining pattern and intensity must be made. If available, computer-assisted image analysis

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Cell Proliferating Antigens 237

can provide an objective and reproducible assessment (Biesterfeld et al., 1995). The pres-ence of Ki-67 antigen can also be determined by flow cytometric methods with improvedreproducibility but at the cost of cell morphology details (Steck and El-Naggar, 1994).

Limitations of Immunohistochemistry

The labeling index (percentage of positively stained nuclei) is often found to varybetween fields within the same tumor specimen because of the heterogeneous distributionof proliferating cells, which can introduce sampling error. Also, the values obtained for thelabeling index may vary among laboratories, depending on storage and handling proce-dures, thus limiting the usefulness of a direct comparison of the labeling index values. Inthis respect, interobserver and intraobserver detection variations in the same laboratoryalso cannot be ignored.

Also note that the proliferation rate of the tissue depends not only on the number ofcells in the cell cycle but also on the time taken to complete a whole cell cycle and onwhether cells undergo programmed cell death. Because the Ki-67 labeling index measuresonly the number of cells that are cycling and gives no indication of the time required forthe cell cycle, a tumor might be proliferating rapidly and still may show a low labelingindex, or be proliferating slowly but remain in stage and so have a high labeling index.In other words, a tumor with many cells in a cycle can be strongly immunostained usingMIB-1 antibody even though the tumor has a slow cell cycle and a low proliferating rate(Jansson and Sun, 1997). In contrast, a tumor with a short cell cycle and high proliferationrate might not be stained since there are few cells in the cycle.

The above-mentioned possibility is one of the reasons for lack of association betweenKi-67 immunostaining and clinicopathological variables and prognosis, for example, incolorectal carcinoma. For this and other reasons several studies have indicated that Ki-67staining has very little prognostic value in gastric and colorectal carcinomas (Victorzon et al.,1997; Jansson and Sun, 1997). However, in spite of these potential limitations, the assess-ment of proliferation using the Ki-67 labeling index provides valuable prognostic informa-tion in many tumor types, including lymphomas, gliomas, and breast tumors. Ki-67 labelingindex is considered to be a more objective way of predicting malignant transformation thantraditional histopathological evaluation alone.

Antibodies

Monoclonal antibodies Ki-67, MIB-1, Ki-S5, and MIB-5 recognize Ki-67 antigenson sections of formalin-fixed and paraffin-embedded tissues. Using these antibodies inconjunction with immunohistochemistry, a rapid and reproducible determination of thegrowth fraction of a given human cell population can be accomplished. The determinationof the growth factor is an objective aid for defining the outcome of an individual tumorcase and is particularly useful for selecting appropriate individual tumor therapy.

The Ki-67 antibody was obtained in studies aimed at the production of monoclonalantibodies to nuclear antigens specific to Hodgkin and Sternberg-Reed cells (Gerdes et al.,1983). There is a highly significant correlation between the mean value of the growth

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fraction determined with this antibody and the histopathological grade of malignancy.Immunostaining of Ki-67 antigen with Ki-67 antibody can be enhanced by exposing thecells to increasing ionic strength (1.15–1.6 M NaCl) during fixation with paraformaldehyde,suggesting increased accessibility of the antibody to the epitope (Bruno et al., 1992). Notethat epitope denaturation increases at higher ionic strengths. This antibody is used mostlyon frozen sections, although it can be employed on sections of fixed and paraffin-embeddedtissues after treatment with an antigen retrieval method such as microwave heating. However,the immunoreactivity of this antibody tends to be inconsistent because the epitope in freshspecimens may be lost during routine histopathological processing.

To circumvent the limitations of Ki-67 antibody, Ki-67 antibody equivalent murineantibodies (MIB-1–3) were generated against bacterially expressed parts of the Ki-67 cDNAcontaining three 62-base-pair repetitive elements encoding for the Ki-67 epitope (Key et al.,1993). MIB-1 shows affinity with both native Ki-67 antigen and recombinant parts of theantigen. MIB-1 has excellent immunostaining properties for Ki-67 antigen, not only infrozen tissues but also in routinely fixed and paraffin-embedded specimens. In fact, MIB-1exhibits an immunostaining pattern identical to that of Ki-67 antibody in fresh specimens.This advantage of MIB-1 allows retrospective studies using archival specimens. The versa-tility of MIB-1 as a marker of Ki-67 antigen in a wide variety of malignant neoplasms isindicated on pages 39 and 239.

Ki-S5 is another antibody generated against the Ki-67 antigen to label a formalin-resistant epitope in routinely processed tissues (Kreipe et al., 1993). Crude nuclear extractsof the Hodgkin-derived cell line L428 were used for the immunization of mice and the pro-duction of this antibody. The immunoreactivity of Ki-S5 antibody is confined to the nucleiof proliferating cells and, unlike Ki-67 antibody, does not cross-react, for instance, withcytoplasmic antigens of epithelial cells (Rudolph et al., 1995). Moreover, Ki-S5 antibodyyields identical results in fresh or fixed tissues. Parallel staining of Ki-67 and Ki-S5 anti-gens using Ki-67 and Ki-S5 antibodies, respectively, yields almost identical results in non-Hodgkin’s lymphoma (Kreipe et al., 1993). Retrospective studies relating the proliferativeactivity to clinical outcome are rendered possible with antibody Ki-S5 using archival spec-imens that have been fixed and embedded. Ki-S5 antibody is available free on request fromthe Institute of Pathology, Kiel, Germany. MIB-5 is yet another antibody that recognizeshuman Ki-67 antigen (Kosco-Vilbois et al., 1997).

Ki-Sl is another IgG mouse monoclonal antibody that was generated by immunizingBALB/C mice with crude nuclear extracts from the human lymphoma cell line U937(Sampson et al., 1992). This antibody recognizes a 160-kDa cell cycle–associated nuclearantigen and can be used on sections of formalin-fixed and paraffin-embedded tissues. It isconsidered useful for prognostic information in breast carcinoma (Sampson et al., 1992).To my knowledge, the antigen recognized with Ki-S 1 is uncharacterized.

Recently, a new monoclonal antibody, MIB-5 (Immunotech, Westbrook, ME), wasgenerated using bacterially expressed parts of the human Ki-67 cDNA (Gerlach et al.,1997). This antibody is equivalent to the prototype antibody Ki-67 but has the additionaladvantage of being able to react with the rodent-equivalent, cell cycle–related nuclearprotein. MIB-5 antibody identifies cycling cells in embryonic and adult rat tissues fixedwith formalin and embedded in paraffin using antigen retrieval with a pressure cooker andimmunohistochernistry. The antibody is effective in both fresh and formalin-fixed tissues,as well as in archival specimens for retrospective studies. MIB-5 antibody should also betried in normal and neoplastic human tissues.

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The aforementioned antibodies recognize different epitopes of the Ki-67 antigen withvariable survivals during fixation and embedding or differential expression during the cellcycle. These antibodies have different affinities for the recognized epitopes and influenceimmunostaining patterns (Mauri et al., 1994). It is known, for example, that although mor-phological and cell cycle distribution of MIB-1 expression is identical to that of Ki-67 anti-body, these two antibodies react with different epitopes of the Ki-67 antigen. Since theseantibodies are not interchangeable with one another, the cutoff values to define high- andlow-proliferating tumors that have been adopted in previous studies with Ki-67 immuno-staining on frozen sections cannot be applied with antibodies such as MIB-1 and Ki-S5.

Recent Applications of MIB-1 Antibody

It is well established that advanced stages of tumor progression are characterized byan increased growth fraction within the neoplastic cell population. The presence of a rele-vant growth fraction is also related to widely accepted prognostic parameters in humanmalignancies (Özer et al., 1999). The Ki-67/MIB-l index indicates the proliferation rate oftumor cells and thus is a potential prognostic factor. In addition to its prognostic relevance,the index provides information on the response to clinical treatment, based on the analysisof retrospective and prospective clinical studies.

Table 10.1 presents recent examples of the usefulness of Ki-67 antigen–MIB-1 antibodycomplex as a marker of cellular proliferation (growth fraction). However, the usefulness ofthis antibody is restricted to certain species and is not applicable, for example, to rat tissues(personal communication, K.-H. Wrobel).

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Ki-67 Antigen Retrieval Using Microwave Heating

Tissue specimens are fixed with 4% formalin for 24 hr and embedded in paraffin.Sections ( thick) are mounted on poly-L-lysine–coated slides, deparaffmized inxylene, rehydrated in a descending series of ethanol, and dried for 1hr at 60°C.Endogenous peroxidase activity is blocked with 1% in methanol for 15 min. Theslides are placed in a plastic jar filled with 0.01 M sodium citrate buffer (pH 6.0), which isplaced in a microwave oven. They are treated at 750 W for three periods of 5 min each.During each heating cycle the buffer level is checked, and the evaporated portion isreplaced with distilled water.

The jar is removed from the oven and allowed to cool for 20 min at room tempera-ture. Following a brief rinse in PBS (pH 7.4), 10% normal rabbit serum is applied for20 min to block nonspecific protein immunostaining. The mouse monoclonal antibodyMIB-1 (Immunotech, Westbrook, ME) is applied overnight at 4°C in a humidified cham-ber. For negative controls, the sections are incubated for the same duration in normalserum in place of MIB-1 antibody. After a rinse in PBS, the sections are incubated in horseantimouse biotinylated antibody (Vector Lab., Burlingame, CA), followed by staining withavidin-biotin complex (Vector Elite ABC) for 1 hr. The peroxidase reaction is developedfor 1 min with 0.05% DAB (Sigma) as chromogen. The sections are counterstained withMayer’s hematoxylin for 1–5 min and mounted in Histomount or gelatin-glycerin.

The Ki-67 labeling index (percentage of Ki-67 positive cells) can be determined byscoring 500–1,000 cells. The labeling index can be performed by ocular micrometry on aLeitz or any other appropriate light microscope by using a total magnification of 400.Immunohistochemical staining reactivity is regarded as positive when the stained cellsoccupy more than 5% of the observed field. Multiple fields of a viable tumor should beexamined to minimize erroneous ratings caused by a focal or regional distribution of theproliferating cells (Fig. 10.3). For example, renal tumors are composed of three histologi-cal components: undifferentiated embryonic cells (blastema) and variably differentiatedepithelial and mesenchymal cells (stroma). Only nuclei with unequivocal reactivity shouldbe scored as positive.

Ki-67 Antigen Retrieval Using Autoclave Treatment

Specimens from a giant-cell tumor of bone are fixed either with 10% buffered forma-lin or 70% ethanol, decalcified with 5% EDTA in 0.1 M cacodylate buffer (pH 7.4) for 7days and embedded in paraffin (Tsuji et al., 1997). Sections ( thick) are placed onpoly-L-lysine–coated slides (Sigma), deparaffinized, and rehydrated. Endogenous peroxi-dase is blocked with 1 % (Sigma) in methanol for 5 min. After being rinsed with dis-tilled water, the sections are placed in glass Coplin jars containing 10 mM sodium citratebuffer (pH is adjusted to 6.0 with 2 N NaOH) and heated in an autoclave for 5 min at 100°C.

Before being removed from the autoclave, the jars are allowed to cool in the autoclaveuntil the temperature has reached 50°C. After the jars have reached a temperature of 30°C,the sections are incubated overnight at 4°C with MIB-1 antibody at a concentration of

The sections are treated successively with biotinylated antimouse IgG anti-body diluted 1:300 for 30 min, streptavidin-biotinylated peroxidase complex, diluted 1:50

Cell Proliferating Antigens 241

for 30 min, and solution prior to counterstaining with hematoxylin. In controls,sections are stained with the conventional ABC method without autoclave treatment.Autoclave heating at 120°C for 5 min results in increased staining but may be accompa-nied by some damage to the cell morphology. Compared with formalin fixation, ethanolfixation results in stronger staining but less than adequate preservation of cell morphology.Both background staining and false-negative staining are absent. Figure 10.1 shows theresults of Ki-67 antigen retrieval using autoclaving.

PROLIFERATING CELL NUCLEAR ANTIGEN

Proliferating cell nuclear antigen (PCNA) is so named because of its initial discoveryas an autoantigen found in the nuclei of proliferating cells (Miyachi et al., 1978). It wasoriginally detected with serum from patients with systemic lupus erythematosus, whichwas found to contain an antibody against a nuclear antigen present in proliferating cells. Itwas subsequently identified as an S-phase protein and named cyclin, but gradually thisterm has been phased out.

Proliferating cell nuclear antigen is a 36-kDa highly evolutionary conserved eukaryotic,acidic protein at both the protein and DNA sequence levels. Crystallographic studies haveshown that PCNA can self-associate as a trimer, forming a hexagonal ring with sixfoldpseudosymmetry and a central hole (Gulbis et al., 1996). In the center of the trimer is acavity that is sufficiently large to accommodate duplex DNA. This cavity is lined with

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positively charged helices facilitating interaction with the negatively charged sugar-phosphate backbone of DNA (Cox, 1997).

The toroidal structure of PCNA has distinct front and back faces that might providea variety of sites for interaction with other proteins. The loop region between the twindomains of each monomer is a highly immunogenic exposed site that is important forinteraction with other proteins. Such functional protein partners are thought to be crucialin regulating the role of PCNA in replication and repair. The aforementioned symmetrycould define the directionality of PCNA movement as it slides along DNA.

The cell cycle is composed of S, and M phases, in addition to a resting phaseduring which cells are quiescent or senescent. The expression of PCNA increases at

the end of the phase immediately preceding DNA synthesis, reaches a maximum duringthe S phase, and declines through phase. It accumulates in larger subnuclear clumps inS phase, which represents matrix-associated replication factories (Cox, 1997). This totalnuclear PCNA is tenaciously associated with the replication sites and is not removed by deter-gents or high salt (Bravo and Macdonald-Bravo, 1987). Although PCNA is present through-out the cell cycle, its levels are almost negligible in long-term mitotically quiescent andsenescent cells, compared with proliferating cells and increases dramatically during mitosis.

Proliferating cell nuclear antigen is involved in DNA replication as well as in DNArepair synthesis. This antigen is required for processive DNA synthesis catalyzed by DNApolymerase delta, which is one of the enzymes vital for DNA replication. Crystallographicstudies show that three PCNA molecules, each containing two topologically identicaldomains, are tightly associated to form a closed ring (Krishna et al., 1994). The dimen-sions and electrostatic properties of the ring suggest that PCNA encircles duplex DNA,providing a DNA-bound platform for the attachment of the polymerase.

Accumulated evidence indicates that PCNA also plays a critical role in the initiationof cell proliferation, and its expression is elevated almost exclusively during the S phaseof the cell cycle. However, not all studies support this observation. This antigen, asdetected by PC 10 antibody, does not accurately reflect the S-phase fraction in gastricmucosa, as determined by bromodeoxyuridine (BrdU) labeling (Lynch et al., 1994).

Available evidence indicates that PCNA is also involved in DNA nucleotide excision-repair. This role is exemplified by the demonstration that PCNA can be found associatedwith chromatin at all phases of the cell cycle after ultraviolet irradiation in vitro (Toschiand Bravo, 1988). Recently it was shown that not only DNA polymerase delta but DNApolymerases beta and epsilon are also involved in the base excision repair subpathways(Dianov et al., 1999). In addition, PCNA may be expressed by noncycling cells in vivowhich are undergoing DNA repair (Hall et al., 1993).

Immunohistochemistry

Preservation of cell morphology and antigenicity is a prerequisite to reliable immuno-histochemistry of PCNA. Optimal fixation is especially important for antigens involved inDNA synthesis and cell proliferation because correct estimation of the proliferating cell frac-tion is necessary for diagnostic, prognostic, and therapeutic purposes. For example, in malig-nant tumors and vascular injury (restenosis), the distinction between quiescent cells and cellsgoing through the cell cycle is important because many therapeutic agents are effective only

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against cells that are proliferating (Mintze et al., 1995). It should be noted that markers suchas PCNA can identify those cells progressing through parts of the cell cycle but can fail todetect significant numbers of slow or extended proliferating cell cycle populations.

Progressively longer fixation with formalin tends to reduce and eventually maydestroy antigenicity. However, in most cases, masked antigens can be unmasked by theantigen retrieval method. Figure 10.4 (Plate 5A, B, C, D) shows the difference betweenoptimally fixed and overfixed tissues in the immunoreactivity of PCNA. Generally,buffered formalin (10%) at pH 7.0 is recommended for fixation for 4hr at room tempera-ture. Although zinc formalin was used for 4–8 hr as a fixative for studying PCNA in pigileum (Mintze et al., 1995), it is not recommended. PCNA retrieval on sections (thick) of formalin-fixed and paraffin-embedded tissues can be obtained by heating in a hotwater bath at 90°C for 2 hr in 0.01 M sodium citrate buffer (pH 6.0). Alternatively, PCNAcan be retrieved by microwave heating (700 W) for two cycles of 5 min each with a 1-mininterval in tissues fixed for any length of time (see Fig. 10.3). This treatment is also effec-tive whether tissues are fixed with formalin or Bouin’s solution. The PC10 and 19A2 anti-bodies are preferred over MAB 424 for PCNA immunohistochemistry, and PC 10 is betterthan 19A2. Table 10.2 shows recent examples of immunohistochemical localization ofPCNA antigen in various carcinomas.

Limitations of PCNA Immunohistochemistry

The reliability of PCNA immunostaining has been questioned (Louis et al., 1991;Harrison et al., 1993; Figge et al., 1992). In fact, some studies have ruled out a prognostic sig-nificance for PCNA expression. The use of PCNA as a reliable marker of cell proliferation,

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for instance in intraoral squamous cell carcinoma of the head and neck, has been ques-tioned (Nylander et al., 1997). In certain cases, PC 10 antibody–immunoreactive cells mayexceed those expected, especially in neoplasia (Hall et al., 1994).

Both the preparatory procedures and endogenous factors play a role in the unpre-dictability of immunohistochemical results of PCNA. A number of factors—includingsample size, fixation, specific epitope involved, type and source of antibody and its con-centration, quality of immunostaining and type of detection system, selection of micro-scopic fields, distinguishing immunopositive nuclei from immunonegative ones, andthe threshold at which a particular staining is termed positive—are responsible for varia-tions in the assessment results. In addition, because PCNA immunoreactivity variesconsiderably within a single tumor specimen, an inexperienced observer can easilymiss the areas active in tumorigenesis, especially when the rate of proliferation is low(Sallinen et al., 1994).

Examples of endogenous factors that may cause lack of reproducibility of PCNAstaining are given below. In some organs normal tissues show high levels of PCNA expres-sion that is not associated with proliferation, i.e., nonproliferating cells also express PCNA(Harrison et al., 1993; Hall et al., 1994). This phenomenon is thought to be due to changesin PCNA regulation in association with neoplasia and the effect of growth factors on tran-scriptional and posttranscriptional processes (Hall et al., 1990). Growth factors can mediatePCNA expression in cells that need not enter the cell cycle. Epidermal growth factor andTGF have been shown to increase PCNA expression in the mouse pancreas, and it has alsobeen demonstrated that tumors can induce PCNA expression in adjacent normal tissuesthat are not proliferating (Hall et al., 1994).

The above-mentioned phenomenon may also be due to the long half-life (~15–20hr)of PCNA. The long half-life allows cells that are no longer in the cell cycle to continue toexhibit PCNA staining (Scott et al., 1991). Using PCNA immunohistochemistry alone it isnot always possible to make a definite distinction between actively proliferating cells andcells arrested in the cell cycle. Thus, arrested cells become a confounding factor.

The reproducibility of PCNA immunostaining analysis can be improved by computer-assisted image analysis (Sallinen et al., 1994). This approach also improves the repro-ducibility of quantitation among observers. The effect of tumor heterogeneity is minimizedthrough this protocol because large tissue areas can be analyzed. Moreover, compared withvisual assessment, computer-assisted analysis is faster. However, even in the computerized

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assessment, interfield variations resulting from tumor heterogeneity are the primary reasonfor the potential lack of reproducibility of the proliferation analysis. Figure 10.3 shows PCNAheterogeneity in breast carcinoma. Considering the conflicting opinions expressed in the lit-erature, the specificity of PCNA (and Ki-67) as a proliferation marker needs to be reassessed.

In light of the above-mentioned limitations, it seems that Ki-67 is a more specificmarker for cell proliferation than PCNA. This suggestion is strengthened by the observa-tion that ependymal cells (which are unable to regenerate) are PCNA positive but Ki-67negative (Funato et al., 1996). In view of the aforementioned and other factors known toinfluence PC 10 labeling of PCNA, it should not be accepted uncritically as a marker of cellproliferation in sections of paraffin-embedded tissues.

Immunostaining of PCNA on Cryostat Sections

Although the following method is carried out without the typical antigen retrievalstep, it is reliable for immunohistochemical detection of PCNA using cryostat sections(Wrobel et al., 1996). Tissues are fixed by vascular perfusion for 15 min with a mixture of50% methanol and 10% paraformaldehyde in 10 mM phosphate buffer, followed by addi-tional fixation by immersion for 1 hr in the same fixative. They are hydrated sequentiallyin 50% and 10% methanol, washed in 0.1M phosphate buffer, and passed through a gradedseries of sucrose solutions (10%, 20%, and 30%). Following immersion in Tissue TEKOCT Compound (Miles, Elkhardt, IN), the specimens are snap-frozen in liquid nitrogen.

Cryostat sections ( thick) are mounted on gelatin/chrome-alum-coated slidesand air-dried for 30 sec. The remaining incubation steps are carried out in a moist cham-ber. The preincubation is carried out for 45 min in the blocking buffer containing 0.1MTris (pH 7.4), 0.15% Thimerosal, 0.8% Triton X-100, 0.8% NaCl, 20% normal goat serum,and 20% fetal calf serum. After rinsing three times for 10 min each in TBS consisting of0.1 M Tris (pH 7.4), 0.8% NaCl, and 0.0015% Triton X-100, the sections are incubatedovernight at room temperature with the primary monoclonal mouse antihumanPCNA/clone PC10 (diluted 1:3000 in PBS) (Oncogene, Uniondale, NY).

The sections are rinsed in TBS as above and incubated for 1 hr in the secondary anti-body goat antimouse/biotinylated IgG (diluted 1:200 in the blocking buffer) (Jackson,West Grove, PA). Following rinsing in TBS, blocking of endogenous peroxidase is accom-plished by treating the sections with 0.002% phenylhydrazine for 10 min and with10% for 20 min. This is followed by rinsing in TBS and incubation for 1 hr in avidin-biotin peroxidase complex (ABC) (Vector, Burlingam, CA). The sections are rinsed inTBS as above and developed with 0.5 mg/ml DAB in 0.1 M Tris (pH 7.4) containing0.002% 0.04% and 0.012% They are rinsed in TBS,dehydrated, and mounted. Controls can be carried out by omitting the primary antibody orsubstituting the primary antiserum with nonimmune serum diluted 1:500 in blockingbuffer. The results of this procedure are shown in Figure 10.5.

P53 ANTIGEN

p53 antigen was discovered before the gene, but both the gene and its protein arecalled p53. The term p53 was originally given to the phosphoprotein of molecular weight

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53 kDa produced by the p53 gene. This 20-kb human gene consisting of 11 exons islocated on the short arm of chromosome 17 in region 17p13.1, and its mutation occursmost frequently in exons 5–9. The exon 5–9 region is highly conserved through evolutionand is presumably of functional importance. Approximately 95% of the reported p53

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mutations have been found in these exons and their intervening introns. However, p53mutations outside exons 5–9 have also been found in human tumors (Greenblatt et al.,1994). In many types of tumors one copy of the short arm of chromosome 17 is often lost.Detailed studies of this chromosome have demonstrated that 17p13.1, which maps the p53gene, is consistently lost in tumors (Baker et al., 1989).

Human p53 protein comprises 393 amino acid residues. It was first detected in SV40transformed cells by virtue of its ability to form a stable complex with the SV40 largeT antigen (Lane and Crawford, 1979). Later it was found that many transformed cell lines,including primary human tumor cells from patients with various types of tumors, con-tained an elevated level of p53, whereas nontransformed cells contained only smallamounts of this protein. The genomic organization of this gene exhibits a striking degreeof similarity in different species (Furihata et al., 1995).

p53 protein contains three main functional domains: an N-terminal acidic transactiva-tion domain, a central DNA-binding core domain, and a C-terminal homooligomerizationdomain (Fig. 10.6). All three domains are required for efficient binding of p53 to recognitionsites within its physiological target genes and for transcriptional activation of these genes.The vast majority of tumor-associated p53 missense mutations occur within the core domain.

More than 95% of the alterations in the p53 gene are point mutations that produce themutant p53 protein, which in most cases has lost its transactivational activity, resulting in lossof tumor suppressor activity. p53 is the most commonly mutated gene in human cancers, andsuch a gene is involved in the development of at least 50% of clinical tumors (Darnton, 1998).

Wild-Type p53 Protein

Normal p53 gene is a critical controller of normal growth and homeostasis of cellsand tissues. It acts as a guardian of the genome by preventing the proliferation of cells with

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damaged nuclear DNA. This is accomplished by the production of normal (wild-type) p53protein under normal physiological conditions. This protein is expressed at low levels andhas a short half-life due to rapid turnover mediated by ubiquitination and proteolysis.Wild-type p53 becomes stabilized and activated in response to a number of stressful stim-uli, including exposure of cells to DNA-damaging agents, hypoxia, nucleotide depletion,or oncogene activation. The activation allows this protein to carry out its function as atumor suppressor through a number of growth-controlling endpoints. These include cellcycle arrest, apoptosis, senescence, differentiation, and antiangiogenesis.

It is apparent from the above discussion that the two primary functions of wild-typep53 protein are growth arrest and apoptosis. These and other functions are elicited by reg-ulating transcription of a number of important genes. In fact, the biochemical activity ofthis protein relies on its ability to bind to specific DNA sequences and to function as a tran-scription factor. In other words, wild-type p53 protein acts on downstream genes to arrestthe cell cycle until the damaged DNA is repaired or to cause apoptosis (programmed celldeath). Apoptosis is an additional, normal mechanism for control of cellular numbers. Theconcentration of wild-type p53 protein rises in cells after DNA damage, causing arrest ofthe cell cycle in the (the first gap) phase and blockage of the cell cycle into the S (DNAsynthesis) phase via p21 protein. This arrest allows time for DNA repair by interaction ofwild-type p53 with downstream activators (Darnton, 1998).

As stated above, the arrest of the cell cycle is related to the activation of a number ofgenes, in particular the WAF1/C1P1 gene that encodes p21 protein. The p21 impedes pro-gression along the cell cycle at the transition, regulating cell proliferation and block-ing DNA replication (E1-Deiry et al., 1994). This role of p21 is related to its ability toinhibit cyclin-dependent kinases and PCNA. Therefore, cells lacking p21 may fail to arrestthe cell cycle in response to DNA damage. It can be logically assumed that lack of p21 isan indicator of tumor aggressiveness and is correlated with p53 positivity because themutated p53 product is unable to activate its effector (Zlotta et al., 1999). Many cancersshow significant association between p53 abnormalities and lack of p21 expression.However, a p21 expression independent from p53 is a common feature in some cancers,such as malignant ovarian epithelial cell (Elbendary et al., 1996). Nevertheless, combinedimmunohistological evaluation of p53 and p21 expression deserves careful considerationin histopathological diagnosis.

p300 protein also functions in the stabilization of p53 and contributes to the p53transactivation function in the growth arrest response to DNA damage (Yuan et al., 1999).Accumulation of p53 is due to its stabilization rather than its increased transcription.Deficiency of p300 results in increased degradation of p53. The N-terminal domain of p53interacts with the C-terminal region of p300. Acetylation of the p53 C-terminal domain byp300 stimulates the DNA binding activity of p53.

Mutant p53 Protein

Tumorigenesis usually proceeds through a series of genetic alterations involvingoncogenes and tumor-suppressor genes, each potentially resulting in clonal outgrowth ofcells through selective growth advantage. The following brief discussion deals only withone of the latter genes, p53. Mutation of this gene results in the synthesis of a mutated p53

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protein with a changed conformation, a longer half-life (increased stability), and disor-dered function in terms of cellular growth. It means that mutation of this gene leads to theloss of the guardianship of the genome, which in turn allows progression of cells withdamaged DNA through the cell cycle. Loss of control of genomic stability is central in thedevelopment of cancer.

Both the loss of normal p53 function and the acquisition of oncogenic functions bythe mutant p53 protein can contribute to tumorigensis. Both activating and inactivatingmutations of the p53 gene can contribute to cancer progression. Inactivation of p53protein is caused by mutations and deletions in the p53 gene or by interactions of the wild-type p53 protein with oncogenic cellular or viral proteins, for instance, in the primary peri-toneal carcinoma (Marchenko and Moll, 1997). Indeed, mutations in the p53 gene occurin high frequency in most of the common types of human cancer. For example, chromo-some 17 in more than 50% of both squamous cell and adenocarcinomas of the esophagusharbors missense point mutations (Sasano et al., 1992). Such mutations encode alteredforms of the p53 protein. Approximately 85% of the mutations are missense mutations,with one amino acid substituted for another and consequent alteration of p53 protein con-formation. Thus, the oncogenic potential of p53 depends on the occurrence of a mutationin its coding sequence.

Overexpression of p53 protein is common in human malignant tumors. Accumulationof this protein is usually the consequence of point mutations. Immunohistochemical analy-ses in many kinds of tumors have demonstrated a good correlation between p53 genemutation and overexpression of p53 protein. Such a correlation has also been detecteddirectly by DNA sequencing (Furihata et al., 1995). This correlation is particularly clearfor colorectal and lung carcinomas. It is well established that overexpression of p53 proteinplays an important role in the progression of cancer. However, overexpression of this pro-tein in certain types of tumors has been reported without evidence of p53 gene mutations.Nevertheless, nuclear staining of the majority of tumor cells accompanied by the absenceof reactivity in surrounding uninvolved tissues or stroma is the most commonly observedpattern characteristic of the presence of a missense p53 mutation.

p73

The p53 gene product is not the only factor that induces cell cycle arrest or pro-grammed cell death (apoptosis). Two other genes, p73 and p63, encode proteins with trans-activation, DNA-binding, and tetramerization domains, and they share considerablehomology with p53. Like p53, these proteins also induce cell cycle arrest and apoptosis.Each of these proteins is comprised of several isoforms. The p73 protein is a structural andfunctional homologue of the p53 protein.

cAbl, a nonreceptor tyrosine kinase, regulates p73 to induce DNA damage–mediatedapoptosis. Under certain conditions such as DNA damage caused by ionizing radiation oran alkylating agent, c-Abl is activated (White and Prives, 1999). The kinase activity ofc-Abl is induced, presumably through the action of the stress-induced ataxia telangiectasia-mutated (ATM) gene product, a component of the DNA-damage checkpoint (Shafman et al.,1997). The ATM protein is a widely expressed member of the protein kinases family withsimilarities to phosphatidylinositol 3-kinases.

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c-Abl binds to p73 in cells, interacting through its SH3 domain with the carboxylterminal homooligomerization domain of p73. cAbl phosphorylates p73 on a tyrosineresidue at position 99 both in in vivo and in cells that have been exposed to ionizing radia-tion (Yuan et al., 1999). Agami et al. (1999) have also reported that p73 is a substrate forthe cAbl kinase, and the ability of c-Abl to phosphorylate p73 is markedly increased by

As a result, p73 is able to participate in the apoptotic response to DNA damage.The above findings define a proapoptotic signaling pathway involving p73 and c-Abl.

Unlike p53, p73 protein levels do not increase following genotoxic stress. Moreover,although c-Abl interacts with p53 in an irradiated cell, it does not phosphorylate p53 butstill contributes to radiation-induced arrest by a p53-dependent mechanism (Yuan et al.,1999). Although p73 is related to p53, p53 alone is the tumor suppressor. p73 protein asyet has not been localized immunohistochemically.

Antibodies

A number of monoclonal and polyclonal antibodies to wild-type p53 and mutant p53antigens are available and are extensively used in clinical and basic research (Table 10.3).The binding sites for these antibodies on p53 molecule have been identified (Fig. 10.6 andTable 10.4). These antibodies have been a major tool in the immunohistochemical detec-tion of p53 antigen, especially in tumor tissues. The antibodies can be used for frozen orparaffin-embedded tissues; many of them can be employed for both types of specimens,particularly when an antigen retrieval method is used (Table 10.2). This method decreasesthe immunohistochemical detection threshold of these and other antigens. Such detectionsrely on the accumulation of these antigens, especially mutant p53 antigen. It should benoted that the threshold-lowering method may detect both wild-type p53 (present in smallamounts) and mutant p53 (present in large amounts) in certain cancer-bearing tissues.Such a possibility has been reported in the esophageal squamosa epithelium (Mandard,

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1998). It means that wild-type p53 may be associated with mutant p53. This hypothesisremains to be confirmed by molecular analysis such as sequence analysis.

These antibodies are directed against different domains or epitopes of p53 protein.The amino acid sequence of this protein is shown in Figure 10.6. These domains are dis-tributed throughout the p53 molecule from the to the COOH-terminal end.Most of these antibodies recognize the linear epitopes located in the amino- or carboxyl-terminal regions of p53 protein (Legros et al., 1994a). Studies of antibodies in the sera ofmice or rabbits hyperimmunized with human p53 have confirmed that most of these anti-bodies recognize specific epitopes located in these domains (Legros et al., 1994b). In otherwords, preferential recognition of amino acid residues 1–95 and carboxyl-terminalresidues 300–393 by the antibodies exists (Schlichtholz et al., 1992, 1994). These domainsof p53 protein are highly exposed and thus readily accessible to antibodies for immuno-histochemical detection. The central region of the protein is thought to be buried in theinterior of the molecule. However, Legros et al. (1994b) have been able to direct eightantibodies against this region and thus define four new epitopes. To my knowledge, theseeight antibodies as yet have not been used for immunohistochemical studies.

Some of the monoclonal antibodies mentioned below recognize different p53 mole-cule conformations. Also, detection of p53 with different antibodies depends on the timeof its synthesis. It has been suggested that the p53 epitope for antibody 1620 remains cryp-tic immediately after synthesis in human keratinocytes and may not be exposed until latein the life of the protein (Spandau, 1994). Furthermore, different conformations of p53may predominate in different differentiation stages of the cell or tissue. In addition,differentiation-specific cellular proteins and other proteins that may bind to p53 may maskepitopes on p53 at various stages of differentiation. For example, heat shock protein 70 isknown to associate with p53 (Hainaut and Milner, 1992).

It is hoped that an understanding of the ability of various anti-p53 monoclonal anti-bodies to recognize different conformations of p53 in cells will aid in the elucidationof the role played by this protein in cell proliferation, cellular aging or senescence, apop-tosis, and gene expression (repressing or stimulating). p53 has been implicated in almostall forms of cell growth stimulation and cell growth inhibition. In addition to the informa-tion on antibodies given below, consult Tables 10.3 and 10.4 and Figure 10.6 for theircharacteristics.

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7.

8.

Monoclonal antibody DO-1 (Oncogene Science, Uniondale, NY) detects bothwild-type and mutant p53 in cell cultures, frozen sections, and paraffin-embeddedsections ( et al., 1992). This antibody binds at the 1–45 amino acid sitenear the N-terminus of the p53 molecule.Monoclonal antibody DO-2 has properties similar to those shown by the DO-1antibody ( et al., 1992).Monoclonal antibody DO-7 (Novacastra, New Castle, UK) has the same proper-ties as those shown by the DO-1 antibody ( et al., 1992). Presently, DO-7 is the most commonly used antibody for detecting p53.Monoclonal antibody DO-11 recognizes two adjacent peptides (38 and 39) inthe PEPSCAN series, localizing the minimum epitope to the 10 amino acids, 181arginine to 190 proline. The epitope for this antibody lies in the central part of themolecule, the conserved domain III at the surface of the mutant p53 antigen. Thisantibody completely fails to immunoprecipitate p53 in the wild-type conformation

et al., 1995).Monoclonal antibody DO-12 reacts only with a single peptide 54 in the serieslocalizing its site to the 15 amino acids 256 threonine to 270 phenylalanine. Thepeptide for this antibody lies exactly between conserved domains IV and V.Antibodies DO-12 and DO-11 cross-react with human and mouse p53 and recog-nize epitopes located in the core part of this protein in areas different from thosecontaining the epitopes for DO-12 and DO-11, which are exposed at the surfaceof p53 protein. None of the epitopes recognized by DO-11, DO-12, and PAb 240antibodies contains sites that frequently mutate in human tumors.Monoclonal antibody DO-13 reacts with peptides 7 and 8 in the LPENNVLSPLseries (epitope on human p53) near the N-terminus. This antibody, like DO-1 anti-body, recognizes conformations of both wild-type and mutant p53 antigens andreacts with human p53 but not with murine p53.Monoclonal antibody DO-14 reacts with peptides 13 and 14 in the EDPG-PDEAPR series within the N-terminal region. This antibody recognizes confor-mations of both wild-type and mutant p53 antigens. It reacts with human p53but not with murine p53. In summary, the epitopes recognized by DO-11, DO-12,DO-13, and DO-14 antibodies are restricted to regions of 10–15 amino acids ofhuman p53.Monoclonal antibody PAb 240 (Oncogene Science, Uniondale, NY) recognizes alinear epitope clearly defined as being within the central core of the mutant p53molecule (Gannon et al., 1990). The epitope for this antibody is cryptic in the activeDNA binding form of wild-type p53 but is exposed at the surface of many mutantp53 proteins as well as of denatured wild-type p53 protein.

PAb 240, DO-11, and DO-12 antibodies do not precipitate all of the mutant p53proteins, suggesting that in some mutant molecules the epitopes remain cryptic.Cryptic epitopes can be exposed by denaturation or through mutations. PAb 240antibody can be used for wild-type and mutant p53 proteins on fresh or paraffin-embedded tissues with the aid of an appropriate antigen retrieval method. BecausePAb 240 reacts with a conformational-dependent epitope in the p53 molecule, thisantibody has helped define the occurrence of different conformational forms of thep53 protein.

Cell Proliferating Antigens 253

9.

10.11.

12.

Monoclonal antibody PAb 1801 (Oncogene Science, Uniondale, NY) recognizesthe N-terminal epitope that is denaturation-resistant.Monoclonal antibody Bp53-12 (BioGenex, San Ramon, CA).Polyclonal antibody CM-1 (BioGenex, San Ramon, CA) was raised against full-length human mutant p53 protein (Midgley et al., 1992).Polyclonal antibody R19 (Biotechnology, Santa Cruz, CA) binds near thecarboxy-terminus of rat p53 (Uberti et al., 1998).

A combination of antibodies has been used in immunohistochemistry. Resnick et al.(1995) have compared the efficacy of immunostaining of accumulated p53 in fresh-frozenas well as formalin-fixed and paraffin-embedded lung and upper aerodigestive tract carci-nomas, using antibodies PAb 1801, DO-7, DO-1, or a 1:1 mixture of PAb 1801 and DO-7(or DO-1), in conjunction with microwave pretreatment. Although these antibodies usedalone yielded good immunostaining, the PAb 1801–DO-7 (or DO-1) mixture showed thestaining of the greatest number of cells. The higher sensitivity of the staining achieved withthe mixture is related to the numerous binding sites on the p53 molecule available to thetwo antibodies. In other words, the cumulative binding of two antibodies exceeds the bind-ing of any one antibody. As indicated in Table 10.3, each antibody has affinity for differ-ent epitopes. A mixture of monoclonal antibodies BaGS-3 (1:80) and BaGS-5 (1:80) hasalso been employed for the detection of T and Tn epitopes on breast adenocarcinoma cells(Wang et al., 1998). However, the use of a mixture of antibodies is not in common use.

Examples of Antibody Dilutions

As an example, optimal dilutions of four antibodies commonly used for p53 proteinin squamous cell carcinomas are given below (Piffko et al., 1995).

CM1 1:2,000DO-7 1:200PAb 240 1:10PAb 1801 1:40

Note that a wide range of dilutions of the same antibody is used, depending on thetissue type and whether or not an antigen retrieval method is used. For example, DO-7 anti-body has been used at a dilution of 1:10 for detecting p53 protein in esophageal carcinoma(Yang et al., 1998), while the same antibody was employed at a dilution of 1:1,000 fordetecting this protein in breast cancer tumors (Daidone et al., 1998). There are many simi-lar examples. Substantial differences in the quality and quantity of immunostaining of p53are found even in the same tissue, depending on the primary antibody and the dilution used.

Immunohistochemistry

Wild-type p53 protein is difficult to detect immunohistochemically in normal cellsbecause of its very short half-life (~20 min) and its presence in minute amounts. However,wild-type p53 protein accumulation can be detected by using antigen retrieval techniques(Dowel and Ogden, 1996; Hall and Lane, 1994). On the other hand, because mutant p53

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Cell Proliferating Antigens 255

protein has a longer half-life (~12 hr), and is present in relatively large amounts, it can bedetected easily with or without antigen retrieval pretreatment (Figs. 10.7 and 10.8, respec-tively). In comparison with formalin fixation, alcohol fixation results in increased stainingof p53 antigen, probably due to easy access of the antigen to the large antibody macro-molecules in the absence of protein crosslinks. Allison and Best (1998) have compared theeffects of alcohol fixation with those of formalin fixation, in conjunction with microwaveheating, on the immunohistochemical demonstration of p53, PCNA, and Ki-67 antigens inoral squamous cell carcinoma. They indicate increased nonspecific staining of p53 antigenstaining in the alcohol-fixed tissues. Similarly, fixed and treated tissues also showed p53antigen staining in unexpected tissue components. Another adverse effect of alcohol fixa-tion is the comparatively poor quality of cell morphology preservation, which becomesapparent at higher magnifications. Therefore, formalin fixation is preferred.

Although a large number of immunohistochemical studies demonstrate that p53 over-expression is positively correlated with proliferation rates in many tumor types (Table 10.5),caution is warranted in the interpretation of such results because the presence of an hetero-geneous population of cells within a tumor specimen is well known. This problem mightbe avoided by using cell lines derived from tumors, thus obtaining a homogeneous sourceof tumor cells. However, such cell lines might acquire mutations absent in the originaltumor. Moreover, part of the positive immunoreactivity could result from an accumulationof wild-type p53 protein. Under certain circumstances, wild-type p53 protein may

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accumulate, probably because of complex formation with oncogenic proteins or as aresponse to DNA damage. Another problem is the presence of p53 staining in thecytoplasm in some cases, the significance of which is debatable (Linden et al., 1994;Bosari et al., 1993; Fisher et al., 1994).

Immunohistochemical detection of wild-type and mutant p53 proteins can be carriedout in fresh-frozen as well as formalin-fixed and paraffin-embedded tissues. This is bestaccomplished by using antigen retrieval with microwave heating or other types of heatingsuch as autoclaving. A number of monoclonal antibodies are commercially available, thecharacteristics and sources of which are listed in Tables 10.3 and 10.4 and on pages 50–51.

However, the validity of immunohistological analyses of p53 expression has beenquestioned. There are several factors, such as the source of antibodies and their cross-reactivity, that are potentially responsible for inconsistent results. Demonstration of p53 innormal cells by immunohistochemistry (and flow cytochemistry) should be confirmed,when possible, by Western blot analysis (Nickels et al., 1997). As a general practice, anunusual staining pattern of p53 should be interpreted with caution.

USE OF MULTIPLE ANTIBODIES FOR LABELING P53 ANTIGEN

An antigen is a highly complex, three-dimensional molecule, and the precise locationof epitopes on or within the antigen in most cases is not known. Furthermore, antigenretrieval methods may unfold the antigen molecule, exposing hidden (buried) epitopes.

Cell Proliferating Antigens 257

These methods may break down crosslinks between the antigen and the surroundingproteins, allowing the epitopes to become accessible due to the mechanism(s) responsiblefor the availability of epitopes for immunohistochemistry—a strategy to ensure that recog-nition of the epitope by the antibody is required. Such a protocol is presented below.

A monoclonal antibody or its clones are directed against a specific amino acidsequence of epitope of the antigen molecule. If such an epitope is not accessible to a givenmonoclonal antibody, the interaction will not occur, resulting in false-negative immuno-staining. To avoid this problem, multiple monoclonal antibodies against the same antigenbut reactive to different amino acid sequences can be used. This can be accomplished byusing more than one antibody simultaneously or separately. The approach almost ensuresthe interaction between at least one of the antibodies and its accessible epitope, resultingin positive immunostaining. It is also possible that more than one antibody in the cocktailof antibodies may interact with more than one epitope. As an example, three antibodiesused in three separate studies for labeling p53 is described below.

Seven monoclonal antibodies and one polyclonal antibody used against p53 antigenare given in Table 10.4. Each of the monoclonal antibodies shows specific affinity for a dif-ferent range of amino acid sequences of the p53 molecule. By using three antibodies sep-arately—DO-7 (for 21–25 amino acid sequence), Pab240 (for 213–217 amino acidsequence), and HR 231 (for 371–380 amino acid sequence)—false-negative immunostain-ing of p53 can be avoided (Tenaud et al., 1994). These three antibodies possess specifici-ties for epitope distributed along the p53 molecule as shown in Figure 10.6.

Wild-Type p53 Antigen Retrieval Using Microwave Heating

The PAb 248 monoclonal antibody recognizes an epitope highly preserved betweenmouse and humans (Rotter et al., 1983), which thus can be used for localizing wild-typep53 antigen in human tissues. Using this antibody, wild-type p53 has been localizedimmunohistochemically in the normal human lymphoid and epithelial cells (Pezella et al.,1994). Sections of paraffin-embedded tissues are processed using standard antigenretrieval with microwave heating and the immunoperoxidase technique.

p53 Antigen Retrieval Using Microwave Heating

Tissues are fixed with 10% neutral phosphate–buffered formalin and embedded inparaffin, and sections ( thick) are mounted on poly-L-lysine-coated slides heated at60°C for 30 min. The sections are deparaffinized in four changes of xylene and then rehy-drated in a descending series of ethanol. Endogenous peroxidase activity is quenched byimmersing the sections in 1% in distilled water for 5 min. The sections are rinsed inthree changes of distilled water, transferred to a moist chamber, and covered with PBS.

The slides are placed in 0.1 M sodium citrate buffer (adjusted to pH 6.0 with NaOH)in a plastic jar, which is transferred into a microwave oven. They are heated at 750 W for17 min, with brief interruptions at 7 and 12 min to replace evaporated volume with distilledwater. The slides are cooled to room temperature in the citrate buffer and transferred toPBS. Nonspecific background staining is blocked by treating the sections with diluted

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horse serum for 15 min at room temperature. After rinsing in PBS, the sectionsare incubated in DO-7 antibody (diluted 1:200 in PBS) in a moist chamber for 1 hr at roomtemperature. They are rinsed in PBS and treated successively with biotinylated horse anti-mouse antiserum and avidin-biotin peroxidase complex for 30 min each. This is followedby treating the sections with a solution of DAB (0.5 mg/ml) containing 0.009% hydrogenperoxide. The intensity of the brown reaction product is enhanced by immersing the sec-tions in 0.125% osmium tetroxide. The sections are lightly counterstained with hematoxylinand successively immersed in acid alcohol, lithium carbonate solution, graded ethanol solu-tions, and xylene. The slides are coverslipped with Permount medium. Figure 10.7 showsthe immunostaining of p53 antigen using microwave heating.

Frozen Section Immunohistochemistry of p53

Tissue specimens are snap-frozen at – 60°C in an isopentane freezing bath (Neslab,Portsmouth, NH) (Resnick et al., 1995). Sections thick) are cut on a cryostat,

Cell Proliferating Antigens 259

mounted on poly-L-lysine-coated slides, and immediately fixed with 100% ethanol at 4°Cfor 10 min, followed by air-drying for ~45 min. The slides are stored at –70°C for 12 hr,thawed to room temperature, and rehydrated in PBS (pH 7.4) for 5–10 min. Nonspecificreactivity is blocked by treating the sections for 15 min at room temperature with dilutedhorse serum ( Vector Mouse Elite ABC kit, Vector Labs, Burlingame, CA). Thisis followed by a blocking procedure for endogenous biotin (BioGenex avidin-biotin block-ing kit, Vector Labs) according to manufacturer’s instructions.

The sections are rinsed in PBS, followed by incubation in PAb 1801 antibody (1:100)for 1 hr in a moist chamber. They are rinsed in PBS and treated successively with biotiny-lated horse antimouse antiserum (30 min) and avidin-biotin-peroxidase complex (30 min)at room temperature, using the Vector Mouse Elite ABC kit. The chromogen, a mixture ofDAB (0.5 mg/ml) and 0.009% is added. The intensity of the brown reaction productis enhanced by immersing the sections in 0.125% solution. The sections are lightlycounterstained with hematoxylin and successively immersed in acid alcohol, lithiumcarbonate solution, graded ethanol solutions, and xylene. The slides are coverslipped withPermount medium. The results of this procedure are shown in Figure 10.9.

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Chapter 11

Estrogens

Endogenous estrogens are 18-carbon steroids and were discovered in the 1920s. They areproduced in the ovaries, adrenals, and stroma of peripheral fat. is the dom-inant estrogen in reproductive-age women. Estrogens bind to members of the nuclearreceptor superfamily. The functions of an estrogen are mediated by specific high-affinityestrogen receptors and located in the target cell nuclei. Estrogens clas-sically exert their effects through the receptor mechanism of action. The estrogen entersthe cells by passive diffusion and binds to the ER. Following a series of activation steps,the estrogen-ER complex, associated with the estrogen responsive element, functions as anenhancer for the estrogen-responsive, element-containing genes.

Estrogen is a key intracellular modulator of the processes involved in differentiation,development, and homeostasis. This hormone produces physiological actions within avariety of target sites in the body and during development by activating a specific receptorprotein. Estrogen plays a crucial role in embryonic and fetal development to influencefemale secondary sexual characteristics, reproductive cycle, fertility, and maintenance ofpregnancy. In addition, estrogen modulates lipid and cholesterol homeostasis in females.The hormone also contributes to the neuroprotection seen in females after traumatic orischemic cerebral insults (Roof and Hall, 2000). This neuroprotection can be partlyexplained by invoking estrogen’s lipid-lowering effect. Estrogen also directly affects theblood vessel wall, microvascular vasomotor tone, and production of vasoactive substances.Several mechanisms are responsible for these effects.

Other putative effects of estrogens include preservation of autoregulatory function, anantioxidant effect, reduction of production and neurotoxicity, reduced excitotoxicity,increased expression of antiapoptotic factor bcl-2, and activation of mitogen-activated pro-tein kinase pathways. Also, there is overwhelming data indicating that estrogens enhancesurvival of neurons both in vitro and in vivo (Green and Simpkins, 2000).

Estrogens are synthesized not only in females but also in males. The synthesis of thishormone by cytochrome P450 aromatase in Leydig and Sertoli cells of the testis is wellknown (Carreau et al., 1999). This cytochrome is also found in the brain, where estrogen isimportant for imprinting male behavior (Beyer, 1999). There is clear evidence that the roleof ER in males is associated with the maintenance of fluid reabsorption in the head of theepididymis (Hess et al., 1997). The loss of ER function in males interferes with the resorp-tive function of efferent ductules, a function that is essential for fertility (Hess, 2000).

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The biological importance of estrogen also becomes clear considering the number ofdisease states associated with altered production of this hormone or abnormalities in themanner in which the cell responds to the biological stimulus provided to the cell by estro-gen. It is well known that estrogen replacement therapy is associated with numerous ben-eficial health effects, including a reduction in risk for cardiovascular disease, decreasedincidence of osteoporosis, and a significant reduction in all-cause mortality (Grady et al.,1992; Hunt et al., 1990). Estrogen’s cardiovascular benefits result from improved profilesas well as favorable effects on the vascular wall. It has been suggested that nuclear factor-kBmay be involved in both early and late stages of the inflammatory-proliferative process ofatherogenesis, and the negative cross-talk between ER and this factor may be a funda-mental mechanism in estrogen’s cardioprotection. Other mechanisms are discussed byHarnish et al. (2000).

Estrogen use is also associated with a number of clinically relevant neurological ben-efits, including increased verbal memory, reduced incidence of Alzheimer’s disease, anddecreased neuronal damage from stroke (Sherwin and Carlson, 1997; Paganini-Hill andHenderson, 1994; Schmidt et al., 1996). In addition, estrogen plays a positive role ininhibiting further progress of Parkinson’s disease (Saunders-Pullman et al., 1999). Thereare several possible explanations for estrogen’s effects on memory and cognition, includ-ing modulation of neurotransmitter function and increased synaptogenesis. The direct neuro-protective role of estrogens, as well as the proven clinical safety of these hormones,suggest that estrogen therapy may be useful in treating neurodegenerative diseases as wellas neurotrauma such as head injury and cerebral ischemia, as mentioned above. While therole of estrogens and their receptors in breast cancer is discussed elsewhere in this chapter,it suffices to indicate that paradoxically, in addition to the initial promotor role of estrogensin breast cancer, they prevent spreading of cancer cells. The protective role of againstcancer progression has also been presented elsewhere in this chapter.

ESTROGEN RECEPTORS

Three isoforms of the estrogen receptors (ER) have been identified, cloned, and char-acterized from several species: and (Green et al., 1986; Kuiper et al., 1996;Hawkins et al., 2000). These receptors are members of a superfamily of genes that consistsof nuclear receptors for diverse hydrophobic ligands such as steroid hormones (estrogens,progestins, glucocorticoids, mineralocorticoids), retinoic acids (vitamin A), vitamin D,prostaglandins, and thyroid hormones. Most members of this family are ligand-dependenttransactivators. After hormone binding and transformation, receptor-ligand complexesinteract with specific hormone response element on target genes, regulating transcription.

When not bound to the hormone, ERs exist in an unactivated, untransformed state(as a monomer) and complex with heat shock proteins. In the estrogen-binding state, thereceptors undergo physico-chemical changes, including phosphorylation at specific serineand tyrosine residues that are accompanied by conformational changes (Arnold et al.,1997). These changes result in the dissociation of heat shock proteins from the activatedcomplex and formation of a 5S homodimer with high affinity for estradiol and DNA. Thetransformed dimer binds to its specific estrogen response element located in the promoterregion of estrogen-responsive genes, regulating their transcriptional activity. Estrogen

Estrogens 263

receptor–estrogen response element interactions are augmented by the binding of coactiva-tors or corepressors that further regulate gene transcriptional activity, without binding directlyto DNA, via interaction with specific transcription factors such as Spl (Porter et al., 1997).

Estrogen receptor was first identified in the 1960s, using binding assays that meas-ured the uptake of radioactive estradiol by cytosolic homogenates of tissue (Jensen andJacobson, 1962). The initial studies focused on rat tissues, but soon attention focused onthe detection of ER in human breast cancers (Jensen et al., 1971). In the 1970s it becameclear that the ER could be detected in 60–80% of human breast cancers and that it couldbe useful in predicting the response to endocrine therapy (McGuire, 1975). After 25 yearsthis statement still holds true.

Because biochemical methods used in the 1970s required large amounts of the tissuefor homogenization, the studies concentrated on breast cancer (Fig. 11.1) rather than onnormal breast (Fig. 11.2/Plate 5E). It was not until the development of antibodies againstER, which would be effective for the fixed tissue of a small size subjected to antigenretrieval, that normal breast tissue began to be analyzed for ER.

Even today, a comparatively small number of studies are available on the ER in normalbreast tissues. Consequently, we know much more about abnormal ER than about normalER. Table 11.1 shows the presence of ER in a wide variety of carcinomas.

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Previously only a single type of ER was known to exist as the mediator of thegenomic effects of estrogen in specific target tissues. Later, cloning of a gene encoding asecond type of estrogen receptor was reported in the mouse, rat, and humans(Kuiper et al., 1996; Vladusic et al., 1998). To distinguish between these two ERs, the ini-tial receptor is termed This development has prompted a reevaluation of the estrogensignaling system.

In mammals, the gene is mapped to the q22-24 band of chromosome 14, whilethe gene is mapped to the long arm of chromosome 6 (Enmark and Gustafsson,

Estrogens 265

1999). In addition, several isoforms of these two subtypes were recently reported. Theseare transcribed either by alternative exon splicing or usage of different promoters of a sin-gle gene (Friend et al., 1995; Chu and Fuller, 1997; Ogawa et al., 1998). Estrogen recep-tor gamma is derived through gene duplication. The existence of multiple forms of thereceptor may explain the pleiotropic actions of estrogens in diverse tissues or species.The discussion in this volume pertains to receptors and and

Estrogen receptor alpha and consist of a hypervariable N-terminal domain thatcontributes to the transactivation function (A/B), a highly conserved central domain respon-sible for specific DNA binding, dimerization, and nuclear localization (C), an estrogen-binding domain (E), a hinge region domain (D), and a domain (F) whose function is notknown (Tonetti and Jordan, 1997).

A high level of homology exists between and especially in the DNA-bindingand estrogen-binding domains. These two ERs can form homodimers with themselves orheterodimers, providing three potential pathways for estrogen signaling. However,and differ in the C-terminal ligand–binding domain and in the N-terminal transacti-vation domain. The difference between ER subtypes in relative ligand binding affinity andtissue distribution explains the selective action of ER agonists and antagonists.

Estrogen Receptor Alpha

The human is a complex genomic unit exhibiting alternative splicing and pro-moter usage in a tissue-specific manner. This observation demonstrates the importance oftranscriptional control in the regulation of expression. However, the mRNA sta-bility is subject to hormonal control, suggesting that the regulation of the expression of thisreceptor may also occur at a posttranscriptional level (Saceda et al., 1989). Note thathuman mRNA has a relatively short half-life of approximately 5 hr in the breast car-cinoma cell line MCF-7 after actinomycin D treatment; actinomycin D is the transcrip-tional inhibitor (Kenealy et al., 2000).

Six functional regions (A–F) are recognized in the molecule, which show dif-ferent degrees of amino acid sequence conservation. Human is comprised of 595amino acids with a molecular weight of 66–70 kDa. Conserved domain organizationresponsible for specific functions of is DNA binding, ligand binding, dimerization,protein binding, and transcriptional activation. The hypervariable A/B domain in theamino-terminal region of exhibits little or no conservation between species. Thisregion contains an activation function, is important for transactivation, and is responsiblefor gene and cell specificity.

Region C corresponds to the DNA binding domain and is responsible for specific bind-ing of the receptor to estrogen response elements located in target genes. Region D is thehinge region, which separates the DNA-binding domain from the ligand-binding domain.This region also facilitates conformational changes in the receptor molecule during activa-tion and is important in receptor dimerization. Region D and the C-terminal portion of regionC contain nuclear localization signals and are responsible for nuclear localization. Region Eis located in the C-terminal portion of the receptor and is responsible for ligand binding. Thisregion contains a second activation function domain, involved in transactivation in conjunc-tion with A/B domain. The exact functional role of region E is not clear, although it may play

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a role in distinguishing between agonist and antagonist binding to the receptor molecule(Montano et al., 1995).

is predominantly expressed in specific tissues, such as breast, uterus, and vagina.The receptor plays a key role in many normal physiological processes, ranging fromfemale sexual development and reproduction to liver, fat, and bone cell metabolism. It isalso involved in the biology of breast cancer and is used clinically as an important prog-nostic factor (Fig. 11.3). Significant amounts of the have been detected in more than60% of human breast cancers. Approximately 70% of the tumors respond toantiestrogen therapy compared with only ~5% of the tumors.

In spite of the usefulness of ER immunohistochemistry in the diagnosis and prognosisof the breast cancer, the published data are not always in agreement. The discordantimmunohistochemical ER results reported in the literature are partly owing to the use of dif-ferent monoclonal antibodies. Generally, different antibodies recognize a specific epitopewithin the domains over the entire length of the ER. For example, the difference betweenthe reactivity of ERID5 monoclonal antibody (which targets an epitope in the A/B region)and H222 monoclonal antibody (which targets an epitope in the E region) observed inbreast tumors is considered to be due to the presence of ER variants (Elias et al., 1995).Also, by developing monoclonal antibodies to specific domains of ER, the presence ofstructurally defective ER in breast tumor has been demonstrated (Traish et al., 1995). Thus,

Estrogens 267

specific monoclonal antibodies can be used to identify dysfunctional ER in breast tumorbiopsies. In fact, the failure of some ER-positive breast tumors to respond to endocrinetherapy, the development of endocrine resistance, and progression in human breast cancerare related to the presence of ER variants.

Caution is warranted in interpreting the status of breast cancer because a monoclonalantibody directed toward a certain ER domain may yield false-negative results if changesin the ER molecule have resulted in the absence or conformational alteration of the domaintargeted. An improved approach to assess the ER status in the breast tumor is to use a panelof monoclonal antibodies that target several distinct epitopes within the ER domains. Thisapproach has been successfully used for determining ER status in 46 primary breast car-cinomas using three monoclonal antibodies (AER 311, ER1D5, and LH2) (Santeusanioet al., 2000). According to this study, in patients with breast tumors negative for these threeantibodies, the disease progressed within 8 years from the diagnosis of the tumor, whereasall patients with tumors positive for all three monoclonal antibodies were alive 13 yearsafter surgery. Considering the importance of ER variants in the immunohistochemistry ofbreast cancer, their characterization and functions are summarized below.

mRNA undergoes alternate splicing, generating transcripts containing single, dou-ble, or multiple exon deletions. The presence of such transcripts in breast cancer cell linesand normal and malignant breast tissue specimens has been described (Leygue et al., 1996).Although the exact function(s) of these splice variants is not established, it is likely that theseproteins differ in activity. Such proteins may differentially modulate the ER signaling path-way in normal tissues. Also, changes in the balance of these transcripts could perturb theER signaling pathway and contribute to tumorigenesis, tumor progression, and response tohormone. Therefore, it is important to qualitatively investigate the difference in the levels andpattern of ER splice variant expression between normal and neoplastic tissues.

Conventionally, the variants are characterized by coamplification with wild-typesequences using reverse transcription polymerase chain reaction (RT-PCR). However, thisapproach focuses on small regions of the known wild-type mRNA. Because of this thresh-old detection, spliced transcripts expressed at low levels may fall below the threshold ofdetection. To avoid this and other limitations of the conventional RT-PCR technique, thetargeted amplification method can be used (Poola et al., 2000). This method involves thetargeted amplification of the alternatively spliced molecules as separate gene populationsusing specific primers designed for the alternative splice junctions, without coamplifica-tion of wild-type molecules.

Estrogen Receptor Beta

Estrogen receptor beta has been cloned from rats, humans, and several otherspecies (Kuiper et al., 1996; Mosselman et al., 1996; Lakaye et al., 1998). Human isexpressed in multiple isoforms with various amino acid numbers. Recent studies documentthat the expressed full-length human is comprised of 530 amino acids (Fuqua et al.,1999). The DNA-binding domains of human and human are highly homolo-gous, approaching 96%, while the ligand-binding domain shows only 59% homology.The N-terminal A/B domain, hinge region, and F domain are distinct in sequence between

and The binds the natural hormone (estradiol) with affinity similar to

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that of However, it should be noted that the differences in the distribution andstructure between these two receptors suggest that the two isoforms have different biologicalactivity.

The is present in the nucleus of a wide range of normal adult human and rattissues, including breast, ovary, fallopian tube, uterus, lung, kidney, brain, heart, prostate,testis, oviduct, adrenal, seminal vesicle, and bladder (Saunders et al., 1997; Taylor andAl-Azzawi, 2000). The presence of and in the normal human breast tissue isshown in Figure 11.2. Specifically, this receptor is located in the epithelial cells in mostmale tissues, including the prostate, the urothelium and muscle layers of the bladder, andthe Sertoli cells in the testis. In the uterus, both and are present in epithelial cellslining the lumen and glands. In the lung, is found in the cells lining the bronchiolesand alveoli and smooth muscle.

Estrogen Receptor Gamma

It has been known for some time that some genomic actions of estrogen cannot beattributed to either or For example, continues to protect againstvascular injury in both and knockout mice (Karas et al., 1999). This evidencesuggests the presence of additional types of estrogen receptors. Recently, the presence ofa third type of estrogen receptor, in a teleost fish, the Atlantic croaker (Micropogoniasundulatus), was reported (Hawkins et al., 2000). This receptor is thought to have arisenthrough gene duplication from early in the teleost lineage. Receptors and arealso present in this vertebrate species. The three ER subtype receptors are genetically dis-tinct and have different distribution patterns in this vertebrate. These three subtypes ofreceptors have distinct functions, at least in the hypothalamus.

Distribution of Estrogen Receptors

is more widely distributed than and when both receptors are present in atissue, the former is predominant. is present in the nucleus of a wide range of normaladult human and rat tissues, including breast, ovary, oviduct, fallopian tube, uterus, prostate,testis, seminal vesicle, bladder, and lung (Saunders et al., 1997; Taylor and Al-Azzawi,2000). Based on mRNA analyses, this receptor is expressed in the central nervous system,cardiovascular system, immune system, and gastrointestinal tract (Gustafsson, 1999). In thelung, is found in cells lining the bronchioles and alveoli and smooth muscle. Moderateto high expression of is found in uterus, testis, pituitary, ovary, kidney, epididymis, andadrenal gland. In the uterus, and are expressed in epithelial cells lining the lumenand glands.

Although distribution is closely related to the expression of in sometissues, the expression of these two receptors does not seem to be linked. Some

cells lack while other cells show both of these receptors, and still other celltypes are and A few examples follow. In the endometrium,both and are present in luminal epithelial cells and the nuclei of stroma cells,while expression is weak or absent in the endometrial glandular epithelia. In the

Estrogens 269

ovary, is present abundantly in multiple cell types, such as granulosa cells in small,medium, and large follicles, theca and corpora lutea, whereas is undetectable in thesecell types (Saunders et al., 1997). Breast and pituitary contain both receptors, whereasprostate shows positive immunoreactivity for but negative for An extensive listof the immunohistochemical distribution of and in adult human tissues is pre-sented by Taylor and Al-Azzawi (2000).

ROLE OF ESTROGEN RECEPTORS IN BREAST CANCER

The ER and the progesterone receptor (PR) belong to the steroid hormone receptorfamily of ligand-inducible transcription factors, which play a key role in the developmentand progression of breast cancer. Although breast is influenced by many hormones andgrowth factors, estrogens play an important role in promoting the proliferation of bothnormal and neoplastic breast epithelium. The influence of estrogens on the proliferativeactivity of mammary epithelial cells is mediated by at least three mechanisms: receptormediation, autocrine/paracrine loop, and negative feedback (Kumar et al., 1987; Huff et al.,1988; Soto and Sonnenschein, 1987). However, these mechanisms have not been preciselydefined as to their role in the normal development and differentiation of the breast or inthe initiation and progression of the neoplastic process. Because normal epithelium con-tains receptors for estrogen and progesterone, the receptor-mediated mechanism is a majorplayer in the hormonal regulation of breast development.

Considerable amounts of ER are present in more than 50% of primary human breastcancers. The presence of ER in primary tumors identifies patients with a lower risk ofrelapse and better overall likelihood of survival. Moreover, response to endocrine therapymostly depends upon the presence of ER and PR, the latter indicating functional ER sig-nal transduction because PR expression is regulated by ER. Consequently, ER determina-tion has become an established procedure in the management of patients with breastcancer. Approximately 50–70% of patients with recurrent disease who had ER-positiveprimary tumors respond to hormonal treatment compared with only ~5% of patients withER-negative tumors, suggesting a strong correlation between the growth of breast tumorsin vivo and the presence of ER. However, the duration of response is limited because ofprogression to an estrogen-independent state of the tumor. In other words, patients withER-positive breast cancer have a more favorable clinical course and prognosis and longerdisease-free intervals than those with ER-negative cancer. Therefore, determination of theER content of breast cancer tissue is indispensable for selecting a regimen of treatmentwhen there is a relapse or for predicting the prognosis.

Some prognostic factors predicting failure of endocrine therapy are known. The pres-ence of epidermal growth factor (EGF) indicates poor prognosis and is correlated with lackof response to endocrine therapy in recurrent breast cancer. It is recognized that expressionof EGF receptor is inversely related to ER expression in malignant breast tumors andbreast cancer cell lines, both at the protein and mRNA levels (Klijn et al., 1992). However,~50% of ER-positive tumors contain EGF receptors. But ER and EGF receptors are rarelyexpressed simultaneously in the same malignant cell. The likely reason is that both receptorsignal pathways become uncoupled during malignant progression. This and other evidencedocuments the heterogeneous nature of primary breast tumors.

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A double immunohistochemical method has been used for determining the expressionpatterns of ER, PR, and EGF receptors in breast biopsies (Van Agthoven et al., 1994). Itwas demonstrated that ER/PR and EGF receptors in breast tumor cells were inverselyrelated at the single cell level. However, the expression of these three receptors in individualnormal luminal cells was not mutually exclusive.

Breast Cancer and Tamoxifen

It is well known that the ER level is important as a prognostic and predictive markerin breast cancer patients. ER status is correlated with endocrine therapy. Tamoxifen is themost common partial antiestrogen and is used for treating all stages of breast cancer.Clinical trials demonstrate that tamoxifen is useful for the treatment of breast cancer(Fisher et al., 1998). It acts by competitively binding to ER, but its activity ranges fromfull estrogen antagonist to a partial agonist in different tissues. Its effectiveness varies withthe prevailing estrogenic environment (Furr and Jordan, 1984).

The effectiveness of tamoxifen can be evaluated in relation to Ki-67 antigen (a nuclearproliferation marker), which is useful in determining the prognosis of breast cancer. (Ki-67antigen is discussed in Chapter 9.) The relationship between ER levels and Ki-67 antigenexpression before and after tamoxifen treatment has been investigated (Dardes et al., 2000).Immunohistochemical studies demonstrate a decreased Ki-67 labeling index after tamox-ifen treatment in ER-positive patients. Patients with down-regulation of ER expression alsoshow decreased Ki-67 labeling index after tamoxifen therapy. This phenomenon may bebased on the ability of tamoxifen to induce apoptosis and reduce the levels of ER as a tran-scription factor (Dardes et al., 2000) This and other studies indicate that short-term(4 weeks) tamoxifen therapy decreases the proliferation of breast cancer in ER-positivebreast tumor specimens. In relation to ER level there is no difference in the Ki-67 labelingindex level between pre- and posttamoxifen treatment of ER-negative patients.

Recent studies suggest that postmenopausal patients older than 50 with ER-negativebreast cancer, who do not respond well to either hormonal therapy with tamoxifen or adju-vant chemotherapy, may have a significant response to vaccination with autologous tumor-associated antigens (Jiang et al., 1999). Such a vaccination results in a reduction in serumIL-6 concentration in patients with ER-negative breast cancers; it is known that estrogenrepresses IL-6 expression. This approach does not have a direct cytotoxic effect on cancercells but is an attempt to promote mechanisms of rejection of the tumor by the host.

ANTIBODIES

Estrogen receptor comprises several structural domains with specific and overlappingfunctions. A number of monoclonal and polyclonal antibodies are available, some of whichshow affinity for specific domains of the ER molecule. The antibodies discussed below areefficient tools for ER immunohistochemistry on sections of formalin-fixed, paraffin-embedded tissues and facilitate the cellular site expression of and receptors inhuman and rat tissues. Most of these antibodies are used for labeling ERs in breast tissuein conjunction with pretreatment with antigen retrieval methods.

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Monoclonal antibody ERID5 (AMAC Laboratories, Westbrook, ME) is produced byusing recombinant human ER (instead of purified ER) for immunization (Al Saati et al.,1993). It detects but not The antibody reacts with the A/B region of theaminoterminal domain of This antibody also reacts with several truncated forms thatare translated from slice variant mRNA, such as those involving Del. 5 and/or Del. 7. Itsaffinity also extends to the 67-kDa polypeptide chain of estrogen obtained by transforma-tion of Escherichia coli and transfection with COS cells with plasmid vectors expressingestrogen.

In comparison with antibody H222, antibody ERID5 is more sensitive, produceshigher nuclear staining intensity, and can be used at a higher dilution (1:100) (Fig. 11.4). Thehigher sensitivity of ERID5 translates into a better correlation with the biological behavior

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of breast cancer than does H222. According to Goulding et al. (1995), however, using theSpearman’s rank correlation method, a highly significant correlation is found between theH scores using antibody ERID5 or antibody H222. For manual immunohistochemistrywith antibody H222, the sections are stained using Abbott’s ER-ICA monoclonal antibodykit. H222 was the first antibody applied to paraffin sections.

Another monoclonal antibody, NCL-ER-6F11, has been generated by using a recom-binant ER protein instead of the peptide antigen approach (Bevitt et al., 1997). Becausemultiple epitopes are presented by the recombinant protein at immunization, productionof a greater number of hybridomas is expected. NCL-ER-6F11 antibody is specific tohuman (see Fig. 11.3) and does not bind to recombinant human NCL-ER-6F11antibody compares favorably with ERID5 antibody, which is also generated usingrecombinant ER.

Mouse monoclonal antibody AER311 (Neomarkers, Fremont, CA), unlike ERID5,recognizes the carboxyl-terminal domain and reacts only with wild-type estrogen, exceptfor DNA-binding truncated protein (Huang et al., 1996). Immunohistochemistry usingERID5 or AER311 can distinguish hormone-binding truncated protein from wild-typeestrogen. The estrogen-enzyme immunoassay method recognizes various mutant proteinsthat can also be detected only by the ERID5 antibody and not by the AER311 antibodybecause these two antibodies recognize different targets on the ER molecule. As statedabove, the AER311 antibody does not react with ERs that lack the hormone-bindingdomain. Immunohistochemical studies using these two antibodies have shown that a num-ber of palpable breast cancers lack the carboxyl terminal in the ER, regardless of wild-typeER mRNA expression (Hori et al., 1999). Other monoclonal antibodies include D75P3,CC4-5 (Ventana Medical Systems) and 6F11 (Vector Laboratories, Burlingame, CA).Compared with CC4-5, 6F11 gives more intense nuclear staining and less cytoplasmicreactivity.

Saunders et al. (1997) have raised a polyclonal antiserum using a peptide specific forThe peptide (CLSKAKRNGGHAPRVLEL) corresponding to amino acids 196-213

of rat was conjugated to keyhole limpet hemocyanin and used to immunize rabbitsaccording to standard procedures. Polyclonal IgGs were purified from serum on a Hitrapprotein A Sepharose column based on the manufacturer’s instruction (Pharmacia).

The monoclonal mouse antibovine (05-394) antibodies directed against SDS-solubilized calf uterus and polyclonal rabbit antirat (06–629) antibodies devel-oped against the N-terminal region of the human sequence are commerciallyavailable (Upstate Biotechnology, Lake Placid, NY). Another polyclonal rabbit antirat

(310) antiserum developed against the C-terminal region of the human sequenceis also commercially available (Affinity Bioreagents Inc., Golden, CO).

To test possible interactions between various ER functional domains, monoclonalantibodies to various regions of the ER have been developed (Traish and Pavao, 1996).Monoclonal antibody F9 was developed against a synthetic 30-mer hybrid oligopeptide.Another monoclonal antibody, NMT-1, was raised against 15-mer peptide from the N-terminal A/B region (amino acids 240–154). Monoclonal antibody 213 was generatedagainst peptide AT3 in the DNA-binding domain (amino acids 247–263).

The effects of binding these site-directed, monoclonal antibodies to specific regionsof the ER molecule on the conformation of this molecule have been determined. Suchstudies indicate that the conformational change within a small stretch of the ER molecule

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caused by the binding of an antibody is transmitted to another distal region of the recep-tor. This phenomenon is exemplified by the binding of the antibody NMT-1 to the A/Bregion of the receptor, which causes the release of the antibody from its epitope in theDNA-binding region. Thus, the binding of these site-directed, monoclonal antibodies tospecific regions of the ER molecule affects the conformation of this molecule.

IMMUNOHISTOCHEMISTRY

Validation of the immunohistochemical method for detecting ERs is through clinicalcorrelation studies. The ultimate usefulness of this methodology depends on its ability topredict clinical outcome, especially the response to hormone therapy. Based on the evalu-ation of the clinical relevance of measuring ER status with immunohistochemistry, there isa statistically significant relationship with clinical outcome. In addition, by comparing theER status measured in the same tumor, using both immunohistochemistry and biochemi-cal ligand-binding assays, 80–90% agreement has been found between these two tests(Clark, 1996).

Positive results with respect to ERs obtained in breast cancer tissue with immunohisto-chemistry is a sign of good prognosis related to higher survival durations. Estrogen andprogesterone receptor levels are frequently negative in malignant tumors with metastases.Positivity thresholds of ERs for both immunohistochemistry and the DCC assay varyamong different published studies. This threshold for immunohistochemistry ranges from5–30% positive nuclei in breast cancer among laboratories. In women, the amount of10 fmol/mg of protein as the cutoff point for defining ER positivity for biochemical ligand-binding assays is accepted worldwide. This amount establishes the relationship betweenthe amount of cytosol protein and the probability of hormone dependence of the tumor.Although the application of immunohistochemistry to benign tumors and malignant tumorsis highly specific, in some cases it may be less sensitive (Martin de les Mulas et al., 2000).

A reliable method of measuring the ER content in human breast cancer is importantfor optimal treatment and a qualified estimate of the recurrence-free survival of the patient.The majority of the studies on the expression of ERs, especially in human tissues,have been accomplished using RNA techniques such as reverse transcription–polymerasechain reaction (RT–PCR) and in situ hybridization. Although the RT-PCR method is aneffective tool to describe the presence of a particular gene in the tissue, this approach doesnot indicate the specific cell that expresses the gene.

In situ hybridization overcomes the problem of cellular localization, but it is difficultto relate the expression of a particular mRNA to the expression of the functional protein.Moreover, this method is difficult to carry out. Immunohistochemistry, on the other hand,is a relatively simple technique that overcomes these problems by identifying the precisecellular localization of the functional protein. This technique, using paraffin sections, pro-vides information on the ER status of tumors very simply and rapidly. In addition, thisapproach is superior to frozen section immunohistochemistry, the dextran-coated charcoalassay (DCC) (see page 276), or the enzyme-linked immunosorbent assay (ELISA) forpredicting the response to endocrine therapy.

It has been demonstrated that a significant enhancement of the predictive power forresponse to tamoxifen on relapse is achieved by immunohistochemical estimation of both

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receptors, ER and PR (PgR) (Barnes and Millis, 1995). Note that although ER or PRnuclear expression alone predicts response to hormonal therapy, combined ER/PR pheno-types have more precise predictive ability than either factor alone. Prognostic and predictivefactors in breast cancer commonly assessed by immunohistochemistry are reviewed byMohsin and Allred (1999). Recently, Taylor and Al-Azzawi (2000) have carried outan extensive immunohistochemical investigation identifying human tissues that express

receptor and comparing the expression pattern of with that ofThe detection of immunohistochemical staining semiquantitatively is preferred over

other methods because by using specific, monoclonal antibodies the ER positive cells areidentified by a distinct nuclear labeling. The staining can range from weak to intense,depending upon the number of recognizable ERs present in the individual nuclei. In fact,some evidence indicates that computerized quantitation is no better than semiquantitativevisual analysis (Schultz et al., 1992; Remmele and Schicketanz, 1993). Also a strong cor-relation has been found between results obtained with true-color, computer-assisted imageanalysis and semiquantitative scoring (Kohlberger et al., 1999).

However, other studies indicate that computerized image analysis is superior to semi-quantitative assessment because of higher accuracy and reproducibility (McClelland et al.,1991). Quantitative immunostaining analysis of ER using the Cell Image Analysis SystemSAMBA 4000 has been carried out (Esteban et al., 1994 a, b, c; see also page 105). Thissystem optimizes measurements, while human deficiencies are reduced to a minimum.Moreover, semiquantitative scoring requires more experience, know-how, and training.Also, ER immunohistochemistry unfortunately has not been subjected to rigorous statisti-cal analysis to define cutoff values associated with clinically meaningful endpoints such asresponse to endocrine therapy. Automated electronic analysis in the near future will estab-lish reliable observer-independent evaluation of immunohistochemical variables. Althoughsome of the computer-assisted image analysis equipment is too expensive for daily, rou-tine use, it is possible to analyze ER and PR expression routinely and inexpensively withgood correlation to clinical outcomes using a relatively inexpensive standard IBM PC andAdobe Photoshop software (Lehr et al., 1997). A similar type of analysis has been carriedout on endometrial samples from patients treated with hormone replacement therapy tohelp predict clinical outcomes (Wahab et al., 1999).

A number of variations of the immunohistochemical methodology are available tolocalize ERs. The expression of these receptors can be studied on paraffin sectionsor frozen sections, with or without antigen retrieval application, although the latter appli-cation is preferred. Various heating treatments, such as microwave heating, autoclave heat-ing, and boiling on a hot plate, are equally effective in unmasking ERs in paraffin sections.An example of the distribution of ERs in the pituitary gland is given below.

Using immunohistochemistry in conjunction with autoclave antigen retrieval, cellulardistribution of and has been accomplished on paraffin sections of fetal and adultrat pituitary glands (Nishihara et al., 2000). The expression of in the fetal pituitary islower than that during the adult period and is limited to the nuclei of anterior lobe cellsfrom day 17 of gestation. In contrast, is present in the nucleus as well as in the cyto-plasm in both the anterior and posterior lobes during the fetal period from day 12 of ges-tation. The distribution of in the adult pituitary is mainly restricted to the anteriorlobe. It seems that plays different roles in the pituitary during the fetal and adult peri-ods. The above evidence also indicates that oncogenetic changes in the expression of these

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receptors are not uncommon. Note that the quantitation of prognostic markers, includingERs, is hampered by a time-related loss of antigenicity, especially in paraffin sections onglass slides (see page 84).

COMPARISON OF IMMUNOHISTOCHEMISTRY WITHBIOCHEMICAL LIGAND-BINDING ASSAYS

Biochemical ligand-binding assays (e.g., dextran-coated charcoal [DCC] assay) weretechnically and clinically validated more than a decade ago. In fact, most of our knowledgeabout the ER in breast cancer prior to the 1990s was obtained by using these assays.However, these tests are more limited than immunohistochemistry; the advantages ofimmunohistochemistry include its being easier to perform, less expensive, safer, and faster.Also, immunohistochemistry is applicable to a wide variety of specimens (e.g., cytologi-cal preparations, frozen tissue blocks, formalin-fixed archival tissue blocks, and paraffin-embedded tissue blocks and sections). This method, in addition, provides direct correlationwith cell morphology.

Another advantage of immunohistochemistry is that tissues of a small size (e.g., biop-sies) can be used. This is important because it is better to detect tumors at an early stage,when they are small. The necessity of early detection cannot be overemphasized. Verysmall tumors and fine-needle aspirates cannot be used for biochemical assays. Althoughthe DCC assay provides quantitative results, it does not take into account the relativeamount of the connective tissue in the specimen, the presence of carcinoma in situ lesions,or normal ducts and lobules. These limitations are not encountered when using paraffinsections. In addition, immunohistochemistry allows the use of archival tissues when freshtissues are not available. This method does not require any special, expensive equipmentand can be carried out in any standard laboratory.

An additional important advantage of immunohistochemical detection of ERs is theprecise histological identification of tissue structures, both tumoral and nontumoral. False-positive results obtained with the DCC method resulting from the presence of remnants ofnormal glandular structures or small dysplastic areas surrounding the tumor can be identi-fied using immunohistochemistry. Also, the DCC assay can yield false-negative resultsbecause the amount of tissue containing the ERs may be too small in the specimen to bedetected. In contrast, immunohistochemistry is able to identify even a tiny tissue contain-ing the ERs. Note that immunohistochemistry measures a fundamentally different propertyof the ER than that obtained with the DCC assay; the former detects the presence of anantigenic epitope, whereas the latter indicates the ability to bind a specific hormone. It ispossible that the hormone binding site may be damaged or nonfunctional, but theimmunoreactive epitope of the ER molecule may still be preserved.

The immunohistochemistry of ERs has been exhaustively compared with the DCCassay. Review of the literature indicates ~85% agreement between these two methods(Allred, 1999). This is true when immunohistochemistry is restricted to fresh-frozen sec-tions. Immunohistochemistry of frozen sections compared with paraffin sections is a morespecific test to detect ER-positive tumors with very low tumor cellularity; the DCC assaygives false-negative results for such tumors. A number of publications have also reportedgood agreement between the DCC assay and immunohistochemistry of paraffin-embedded

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breast cancer tumors and cytological preparations (Allred et al., 1990; Masood, 1989;Shimada et al., 1985). However, immunohistochemistry of paraffin sections is both lesssensitive and reproducible compared with frozen-section immunostaining. One of the dis-advantages of frozen sections is poor preservation of cell morphology. For this and otherreasons, the use of paraffin sections has become more popular than frozen sections.

Note that controversies over the technical and clinical validation of immunohisto-chemistry have not been completely resolved. Whether or not this method should com-pletely replace biochemical ligand-binding assays remains controversial. Despite thiscautionary statement, it is true that the specificity of immunohistochemistry is theoreticallyvalid because it is based on the use of well-characterized monoclonal antibodies raisedagainst epitopes restricted to the ERs.

Dextran-Coated Charcoal Assay

The dextran-coated charcoal (DCC) assay measures the hormone-binding capacity of thecytosol fraction of the tumor tissue (Lee and Chan, 1994). Fresh tissue (more than 100 mg) isimmediately frozen, homogenized, and the cytosol fraction is extracted. Aliquots of thecytosol are incubated with radiolabeled estradiol with and without 100-fold molarexcess of nonradiolabeled competitor (diethylstilbestrol) to displace radiolabeled steroidfrom the low-capacity specific binding sites (ER), but not from the high-capacity nonspecificbinding sites in the cytosol. The unbound steroid is removed by selective adsorption on DCC.

The receptor binding capacity, reported in femtomoles of radiolabeled steroid bound permilligram of cytosol protein, is calculated by subtracting the level of nonspecific cytosol-bound radioligand (radiolabeled steroid with excess of competitor) from the totalcytosol-bound radioligand (radiolabeled steroid without competitor).

SEMIQUANTITATIVE ASSESSMENT OF ESTROGEN RECEPTORS

Two methods are available to determine the estrogen (and progesterone) status in thetissue samples: biochemical assays (e.g., dextran-coated charcoal [DCC] method) andimmunohistochemistry. Immunohistochemistry is very useful in estimating prognosis andmonitoring therapy in breast cancer. An advantage of immunohistochemistry over theDCC assay is that the former can be used with small tissue samples, and it also facilitatesmorphological evaluation of individual tumor cells. In contrast, because ligand-bindingassays only detect estrogen receptors in an unoccupied form, they are prone to interferenceby other endogenous hormones and may give rise to unreliable results. Furthermore, suchassays preclude assessment of estrogen receptor contents of individual cells. Scoring canbe performed according to the two different semiquantitative methods of Remmele andStegner (1986) and Reiner et al. (1987). Specific staining is identified by distinct coloredstaining of nuclei with the histoscore system, which considers both intensity of stainingand percentage of stained cells.

According to the method of Remmele and Stegner (1986), intensity is graded from0–3, where 0 = no staining, 1 = weak staining, 2 = moderate staining, and 3 = strongstaining. The percentage of stained cells is categorized as follows: 0 = 0%, 1 = 1–10% ,

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2 = 11–33%, 3 = 34–55%, and 4 = 67-100%. The two values obtained from the stainingintensities and percentage of positive cells are multiplied. This method results in values of0 (negative) and 1, 2, 3, 4, 6, 8, 9, or 12 (positive).

On the other hand, according to the second system (Reiner et al., 1987), the score isacquired by adding the two values (staining intensities and percentage of positive cells)and obtaining the values 0 (negative), 2 and 3 (low positive), 4 and 5 (intermediate posi-tive), or 6 and 7 (high positive). In contrast to the complex Remmele-score, the Reinerscore is relatively simple and easily applicable, making it more advantageous in daily rou-tine investigations (Biesterfeld et al., 1996). Moreover, the Reiner score has better repro-ducibility than the Remmele score. It is emphasized that at least three different fields on aslide are observed, and 100 cells are counted in each field. More than one observer shouldparticipate independently in the scoring in a blind test. Following scoring, a consensusscore should be established among the observers.

Note that immunohistochemical reactions do not always yield homogeneous resultsbut are subject to variations from slide to slide and from case to case. Because tumors areheterogeneous, caution is warranted in assuming that a particular section of the tumor isrepresentative of the whole tumor (see page 14 for discussion on tumor heterogeneity).

IMMUNOSTAINING OF ESTROGEN RECEPTORS IN PROSTATE TISSUE

Sections ( thick) of formalin-fixed and paraffin-embedded tissues are deparaf-finized with xylene and rehydrated with ethanol (Bonkhoff et al., 1999). Endogenousperoxidase is blocked by treating the sections with 0.3% hydrogen peroxide. The sectionsare placed in 10 mM citrate buffer (pH 6.0) and heated in a microwave oven at 750 W for5 min followed by at 450 W for 5 min. They are washed in PBS and treated for 30 min withnormal rabbit serum. After being washed in PBS, the sections are incubated overnight inmouse monoclonal antibody NCL-ER-6F11, diluted 1:200 (Novocastra) in a humid cham-ber. This antibody is obtained by recombinant protein preparation from MCF-7 cells andis directed against the full-length molecule.

Following rinsing in PBS, the sections are incubated for 30 min in the secondarybiotinylated rabbit antimouse immunoglobulin (Dako). This is followed by applying thehorseradish peroxidase–labeled avidin-biotin complex (ABC-HRP) method according tothe manufacturer’s instructions (Dako). To amplify the signal and enhance immunodetec-tion of the estrogen, the biotinylated tyramine method is applied. After precipitation of thebiotinylated tyramine for 10 min through the enzymatic action of horseradish peroxidaseand hydrogen peroxide (0.1%), the biotin precipitate is detected with an additional appli-cation of the horseradish-labeled avidin biotin complex (Dako) for 30 min in a humidchamber. The peroxidase reaction is developed with DAB (Sigma) to form a brown end-product. Negative controls are performed on consecutive sections by replacing the primaryantibody with a nonimmune mouse serum. Figure 11.5 (Plate 5F) shows staining ofusing NCL-ER-6F11 antibody.

To localize on paraffin sections, 65-kDa antirat ER antibodies are used (UpstateBiotechnology, Lake Placid, NY). This antibody is obtained by immunizing rabbits withsynthetic peptides representing the N-terminal amino acid residues 46–63 of humanThe deparaffinized sections on slides are placed in the Target Retrieval Solution (pH 6.1)

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(Dako) and heated in a microwave oven as described above. After incubation in normalswine serum (Dako) for 30 min, the sections are incubated in the 65-kDa antirat estrogen

antibodies, diluted 1:100 with PBS for 12hr. Detection is achieved as describedabove, except that the secondary biotinylated rabbit antimouse immunoglobulin isreplaced with the biotinylated swine antirabbit antibody (Dako).

To localize in frozen sections, antigen retrieval with heating is not required.210-180-C050 antibodies (Alexis Corporation, Nottingham, UK) are obtained by immu-nizing rabbits with synthetic peptides representing the C-terminal amino acid residues467–485 of human estrogen.

IMMUNOSTAINING OF ESTROGEN AND PROGESTERONERECEPTORS IN FINE-NEEDLE ASPIRATES OF BREAST

The ThinPrep smear with microwave antigen retrieval pretreatment is a reliablemethod for estrogen (and progesterone) analysis in breast carcinoma (Leung and Bédard,1999). The smears are prepared from fine-needle aspirates of patients with breast carci-noma according to the directions in the Operator’s Manual (Cytyc, Boxborough, MA). Thesmears are postfixed for 5 min with Surgipath cytology fixative (Surgical Medical Industries,Richmond, IL) and kept frozen at —70°C until the antigen retrieval step. Silanized-coatedslides are used for better adhesion of cells.

Target Retrieval Solution (Dako S1700) is diluted 1:10 with distilled water andadjusted to pH 6.0. ThinPrep smears are rehydrated in 95% ethanol and distilled water for5 min each. The smears are placed in a plastic slide container with sufficient retrieval solu-tion. The container is immersed in a sufficient quantity of warm water inside themicrowave pressure cooker (Dako, DO300X) and heated in a microwave oven for 20 minat full power until steam is generated. The smears, along with retrieval solution, areallowed to cool for 10 min. The smears are removed and rinsed with warm water, followed

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by Tris-saline buffer (TSB). Immunostaining is performed using the Dako K1900 ER/PRstaining kit (Dako Corporation, Carpinteria, CA). It includes all ready-to-use reagents,antibodies, and detection system. The smears are covered with hydrogen peroxide for5 min, then with distilled water, and then placed in TSB for 5 min. They are incubated inprimary monoclonal antibodies (Dako K1900) for 30 min and then washed with TSB for5 min. This is followed by incubation with biotinylated linking antibody for 10 min andwashing in TSB for 5 min. The smears are incubated with strepavidin-peroxidase for10 min and then washed with TSB for 5 min. They are treated with chromogen substrateDAB for 10 min and rinsed with tap water for 5 min. Counterstaining is accomplished withhematoxylin for 20 sec. The smears are dehydrated, cleared and mounted. The results ofthis procedure are shown in Figure 11.6.

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Chapter 12

Her-2 (c-erbB-2) Oncoprotein

The HER-2/neu oncogene plays a key role in breast and many other cancers. Methods,including immunohistochemistry, are available to analyze tumor HER-2 status (Fig. 12.1).Elucidation of the human genome is expected to significantly improve many aspects ofhealth care, especially the diagnosis and management of cancer. Genetic discoveries arealready resulting in the development of specific, effective, and less toxic cancer drugs thanmany of those in current use. An important example of a less toxic reagent is Herceptin,which targets HER-2/neu gene product, p185 (HER-2).

HER-2/NEU ONCOGENE

HER-2/neu gene was first discovered as a transforming oncogene in a series of ethylnitrosourea–induced rat neuroblastomas, where it was called neu (Schecter et al., 1984).Approximately 15 years ago, this oncogene was isolated independently by two separategroups, which named it HER-2 (Coussens et al., 1985) and c-erbB-2 (Semba et al., 1985),respectively. Further evidence revealed that the two genes were the same (Schechter et al.,1985), and it was renamed HER-2/neu.

The gene is located on chromosome 17 at q21 and encodes a 185-kD glycoproteincomposed of cytoplasmic, transmembrane, and extracellular domains. This tyrosine kinaseglycoprotein has 40% sequence homology to the epidermal growth factor receptor (EGFR)but is distinct from the EGFR. A physiological ligand for this presumed receptor remainsunidentified. In other words, the kinase may function as a cellular receptor for an undis-covered ligand; however, it has been suggested that 17-estradiol may mimic ligand activityfor this oncogene protein (Matsuda et al., 1993).

Amplification and overexpression of the HER-2/neu gene has been reported in a widevariety of tumor types, predominantly of epithelial origin, including those of female andmale breasts (Slamon et al., 1989; Fox et al., 1991), ovary (Slamon et al., 1989; Hellströmet al., 2001), colon (D’Emilia et al., 1989), pancreas (Thybusch-Bernhardt et al., 2001),stomach (Jain et al., 1991), salivary gland (Kernohan et al., 1991), bladder (transitional cellcarcinoma) (Wood et al., 1991), head and neck (Riviere et al., 1991), and prostate(Schwaab et al., 2001). This gene has also been reported in malignant cartilage (Wrba et al.,1988), papillary thyroid carcinoma, and uterine cervical carcinoma (Hale et al., 1992).HER-2/neu has also been implicated in pulmonary carcinomas (Hirashima et al., 2001),

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colorectal adenocarcinomas (Koeppen et al., 2001), skin cancer (Krähn et al., 2001), andlung cancer (Cox et al., 2001).

The amplification of the HER-2/neu oncogene is thought to have significance in theearly stages of malignant transformation, especially in the breast and ovary, and thus may beconsidered a prognostic marker of these carcinomas. A statistically significant correlationbetween the amplification of this gene and survival of patients with breast and ovariantumors has been reported (Slamon et al., 1989; Carr et al., 2000). Because the gene is over-expressed in breast cancer patients, determination of its prognostic significance is beingevaluated. This oncogene is indeed an independent prognostic indicator of a subset of breastcancers that are at high risk of early recurrence, regardless of tumor grade, estrogen/prog-esterone receptor status, or lymph node status (Carr et al., 2000). However, according toSlamon et al. (1987), association of HER-2/neu amplification and poor prognosis isstronger for patients with lymph node metastases than without lymph node involvement.

The HER-2/neu gene is overexpressed in 25–30% of human breast cancers, and in~90–95% of these cases, the overexpression is a direct result of gene amplification. Theamplification or overexpression of this gene is associated with shorter disease-free survivalas well as overall survival. Appropriate follow-up studies have confirmed the prognosticassociation between the HER-2/neu gene alteration and clinical outcome for both node-negative and node-positive breast cancers (Press et al., 1997)

The HER-2/neu gene is also overexpressed in epithelial ovarian cancer. Several studieshave reported that overexpression of this gene is associated with poor survival rates in

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advanced epithelial ovarian cancer (Berchuck et al., 1990; Bast et al., 1992). Anreder et al.(1999) have also reported poor survival rates for patients showing overexpression of theoncogene in the ovarian surface epithelial tumors.

Note, however, that some types of primary tumors, such as breast cancer, may containelevated p185 protein or HER-2 mRNA levels in the absence of detectable gene amplifica-tion, suggesting that in some cases gene amplification is not strictly required for p185expression. In other words, the presence of this protein in some cancer cells itself is an indexof tumor aggressiveness, regardless of HER-2/neu amplification (Dati et al, 1990).

It should be noted that although HER-2/neu gene is considered to be the target genefor amplification at chromosome bands 17ql2–q21, the amplicon harbors several closelylocated genes such as retinoic acid MLNs 50, 51, 62, and 64, gastrin,

hormone and topoisomerase (Järvinen et al., 1999). Manybreast tumors with HER-2/neu amplification show simultaneous amplification or deletionof topoisomerase gene. Amplification of the HER-2/neu is followed by complex sec-ondary genetic aberrations, which lead to amplification or deletion of the topoisomerasegene in a majority of tumors. A simple molecular mechanism suggested for thisphenomenon is that the amplification of the chromosomal segment includes both genes. Theimportance of this information is that the gene copy number aberrations of topoisomerase

may divide HER-2-amplified breast tumors into clinically meaningful entities.

HER-2 ONCOPROTEIN

Her-2/neu is a protooncogene encoding a 185 kDa protein (HER-2). HER-2 is one ofthe epidermal growth factor receptor (EGFR) family of four closely related transmembranegrowth factor tyrosine kinase receptors. These are designated HER-1 to HER-4 (c-erbB-1to c-erbB-4), and they exhibit a high degree of homology to each other. TransmembraneHER molecules exist as inactive monomers on the cell surface but form receptor dimersthat are stabilized by ligand binding to their extracellular domain domain).Dimerization can occur between the same receptor (a homodimer) (Alroy and Yarden,1997). The resulting phosphorylation of tyrosine residues initiates complex signalingpathways that ultimately lead to cell division.

The interaction between the HER monomers and various ligands (e.g., epidermalgrowth factor and transforming growth factor), and the ensuing diversity of signal transduc-tion from the intracellular tyrosine kinase domain, results in a complexity that explains thekey role played by this type I growth factor receptor family in regulating cell growth and dif-ferentiation (van de Vrjver, 2001). It is possible that the function of HER-2 is to stimulategrowth after the formation of heterodimers with other members of the HER family. In fact,a class of ligands, neuregulins, bind to HER-3 and HER-4, causing heterodimerization withHER-2 (Carraway et al., 1994). HER-2 is the signaling subunit and has no independent lig-and. Antibody blockade of HER-2 prevents heterodimerization, eliminating neuregulin-stimulated signaling. The inhibition of this signal may induce tumor regression and delayreturn to a tumor growth phase in patients with aggressive breast cancer.

It is known that HER-2 signaling enhances metastasis in breast cancer cells by inducingendothelial cell retraction, a process that appears to precede endothelial transmigration(Carter et al., 2001). In other words, breakdown of the vascular barrier caused by HER-2

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signaling may be sufficient to facilitate tumor cell transmigration and metastasis. Themolecular mechanism(s) of HER-2-induced endothelial cell retraction is unknown.

It is interesting to note that expression and secretion of an aberrant HER-2 splicevariant has been reported in various cell lines and tissues, which can interfere with theoncogenic HER-2 activity (Aigner et al., 2001). Expression of this truncated 100-kDaHER-2 variant encodes the extracellular domain of HER-2 and inhibits growthfactor–mediated tumor cell proliferation. The exact role played by this variant during theprogression of human cancer is not clear.

HER-2 OVEREXPRESSION

Overexpression of the HER-2 protein is one of the most studied molecular changesthat occur in human cancer. The main cause of HER-2 overexpression is HER-2/neu geneamplification, although protein overexpression has been observed in some subsets of breastcarcinoma despite a lack of gene amplification (Persons et al., 1997). It is reasonable tosuggest that HER-2 may not have an identical role in all tumors. It is known that HER-2overexpression is able to transform cells in vitro, although catalytic activation of this recep-tor may also be involved in other functions. In fact, the precise functional significance ofthis phenotype is uncertain. This uncertainty is reinforced by evidence that HER-2 overex-pression shows stronger association with preinvasive rather than with advanced disease.Moreover, extensive apoptosis occurs in preinvasive diseases. This and other evidenceraises doubts about the precise significance of the HER-2 phenotype in vivo.

Published immunohistochemical studies of overexpression of HER-2 report a widerange of overexpression rates, ranging from 9 to 60%. The likely reason for this variation isthe use of different antibodies, tissue types, and fixation and staining protocols. In addition,different scoring systems result in interpretation differences. To minimize these variationsthe use of a uniform immunohistochemical method, such as the HercepTest, is recom-mended. The HercepTest kit provides standardized procedure and evaluation criteria.

SIMULTANEOUS OVEREXPRESSION OF HER-2 AND P53

Among molecular markers for sporadic breast cancer, p53 protein and HER-2 recep-tor protein have retained special attention. Genetic alterations resulting in overexpressionof these two proteins are a frequent finding in early stages of invasive ductal carcinomasof the breast, suggesting a role in breast cancer pathogenesis and possibly a function indisease progression.

The detrimental consequence of simultaneous expression of these two proteins hasbeen found even in node-negative mammary carcinomas (Albanell et al., 1996). Moreover,HER-2 and p53 overexpression individually correlates with a higher growth rate in mam-mary carcinomas. More recently, Rudolph et al. (2001) also provided evidence that over-expression of either HER-2 or p53 resulted in an increased cycling ratio, which is thoughtto be additive in tumors overexpressing both proteins. p53 expression is significantly moreprevalent in young patients, whereas HER-2 expression shows only a trend toward a higheroccurrence in the young.

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DISTRIBUTION OF HER-2 IN CARCINOMAS

In addition to breast cancer, HER-2/neu gene amplification is common in ovarian,prostate, and gastric cancers, while a relatively low incidence of amplification is found ina large number of cancers arising in other organs, which are summarized below. The asso-ciation of HER-2 and the prognosis of breast cancer patients have been studied most exten-sively. The amplification of this gene and overexpression of HER-2 oncoprotein havebeen found to be associated with poor clinical outcome. However, whether HER-2 is anindependent prognostic factor is controversial.

Astrocytic Tumors

It is difficult to distinguish between benign and malignant astrocytic tumors. Some low-grade astrocytomas behave similarly to anaplastic ones. This problem may be solved throughdetermining the proliferative index of antigens such as HER-2, p53, PCNA, p21, EGF, andEGFR. Such an immunohistochemical study has been recently carried out by Bian et al.(2000). This study indicates that aberrations of HER-2, p21, EGF, and EGFR might be earlyevents in the initiation and progression of astrocytomas, but they are unrelated to histologi-cal grade and prognosis of astrocytomas. On the other hand, p53 overexpression is involvedin all the stages, while PCNA might be important in evaluating astrocytoma malignancy.In this study, sections were pretreated with 0.1% trypsin containing 0.1% prior totreatment with 0.3% in methanol to block endogenous peroxidase.

Bladder Carcinoma

Wood et al. (1991) have used the Southern hybridization method for detecting DNAamplification and a possible structural rearrangement of the HER-2/neu oncogene in 1 of 12bladder tumors. Amplification of this oncogene in the tumor was sixfold that of oncogenefound in placental DNA. Approximately 36% of the tumors studied overexpressed HER-2mRNA, which was 3- to 38-fold that of normal urothelium. HER-2 overexpression occurredin superficial and invasive tumors. Deoxyribonucleic acid amplification occurs infrequentlyin bladder carcinoma, in contrast to its occurrence in some other carcinomas.Immunohistochemical analysis has shown that p185 HER-2 polyclonal antibody is specificfor HER-2 protein overexpression in bladder carcinoma. This study was carried out prior tothe use of Herceptin.

Ewing's Sarcoma

Ewing’s sarcoma is the second most common malignant bone tumor in children. Themajority of patients have microscopic metastases at diagnosis; the lung is the most com-mon metastatic site. This sarcoma is a relatively rare disease with limited therapeuticoptions. The majority of patients are initially responsive to chemotherapy with vincristine,doxorubicin (Adriamycin), and cyclophosphamide. However, relapsed disease is usuallyextremely difficult to treat because of its resistance to chemotherapy (Zhou et al., 2001).

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Clinical studies indicate that overexpression of HER-2 correlates with poor prognosisand shorter patient survival in osteosarcoma (Onda et al., 1995). There is a close associa-tion between HER-2 overexpression and resistance to chemotherapeutic agents (Tsai et al.,1995). Zhou et al. (2001) have investigated HER-2 expression in three different humanEwing’s sarcoma cell lines (TC71, RD, and A4573). It was found that this protein is over-expressed in the three cell lines. This study also indicates that E1A gene therapy may pro-vide a new approach for treatment of this carcinoma. It has been demonstrated thattransduction of TC71 cells with the ElA gene using an adenoviral vector downregulatesHER-2 overexpression in these cell lines (Zhou et al., 2001). Downregulation of HER-2increases apoptosis in tumor cells that overexpress the oncogene.

Intrahepatic Cholangiocellular Carcinoma

Immunohistochemistry has been used for investigating the expression of HER-2-4 in38 intrahepatic cholangiocellular carcinomas (Ito et al., 2001). HER-2 expression wasobserved in more than 50% of the cases but was not related to any clinicopathological fea-tures. HER-3 expression was linked to lymph node metastasis, and HER-4 expression wasdirectly related to proliferating activity and lymph node metastasis.

Laryngeal Squamous Cell Carcinoma

Preembedding immunoelectron microscopy has been used for localizing HER-2 atthe single cell level in laryngeal squamous cell carcinoma (Grzanka et al., 2000). HER-2was located both on the plasma membrane and in the cytoplasm of cancer cells in approx-imately half of the cases investigated (a total of 15 surgical specimens). The polyclonalantibody, rabbit antihuman c-erbB-2 (Dako), was used in this study, which recognizesHER-2 protein on the membrane as well as in the cytoplasm. Membrane and cytoplasmicstaining of HER-2 has also been reported in gastric carcinoma (Lee et al., 1994).According to Grzanka et al. (2000), a significant correlation between HER-2 in laryngealsquamous cell carcinoma and pathological characteristics, such as nodal status andhistological grade, was not found.

Non-Small-Cell Lung Carcinoma

In addition to carcinomas in other organs, HER-2 oncoprotein is overexpressed incancers of the lung. Lung cancer is currently the leading cause of cancer-related death. Mostpatients have inoperable tumors at the time of diagnosis, and metastatic relapse in patientswith operable non-small-cell lung carcinoma remains a frequent event. Genetic abnormali-ties in lung cancer frequently include the mutation, rearrangement, or overexpression of sev-eral genes and their protein products, such as HER-2, p53, and K-ras. The HER-2 protein isstrongly stained immunohistochemically on the cell membrane of adenocarcinoma, and theoverexpression of this protein relative to that of normal alveolar lung tissue is associatedwith a worse prognosis. A soluble form of the HER-2 protein is also found in serum.

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It is interesting to note that some HLA-class 1 antigens confer resistance to theprogression of bronchogenic carcinoma. There is also a relationship between survival timeand HLA for epidermoid lung carcinoma (Prazak et al., 1990). Also, an associationbetween HLA-DR7 and resistance to lung cancer has been reported (Romano et al., 1991).Analysis of this relationship is important for determining not only the etiology but also theappropriate therapy and prognosis of lung cancer. Recently, Yoshimura et al. (2000)carried out a detailed study of this relationship in lung cancer. This study suggests thatHER-2 is correlated with prognostic factors for lung cancer independent of HLA-associatedgenetic factors.

Immunohistochemical studies demonstrate overexpression of HER-2 protein innon-small-cell lung carcinoma (NSCLC). The protein is located predominantly onthe plasma membrane, although its location has been reported in the cytoplasm, especiallyin adenocarcinoma. In a recent immunohistochemical and fluorescence in situ hybridiza-tion study, with the exception of occasional single tumor cells, cytoplasmic HER-2 expres-sion was not seen (Cox et al., 2001), in contrast to some other studies employing differentantibodies and other processing parameters (Tateishi et al., 1991).

In lung cancer, overexpression of HER-2 is associated with a poor prognosis. Unlikebreast carcinoma, HER-2/neu gene amplification in NSCLC is uncommon. However, inadvanced lung carcinoma, the Herceptin is positive. Thus, Herceptin may have a role in thetreatment of a proportion of patients with this disease, but it is of a limited clinical value,especially in the adjuvant setting.

Ovarian Carcinoma

Approximately 25% of primary ovarian carcinoma expresses HER-2 protein, butunlike its expression in breast cancer, it is controversial to what extent HER-2 amplifica-tion and overexpression correlate with prognosis. However, HER-2 expression is more fre-quent in ovarian carcinomas relapsing after chemotherapy (Meden et al., 1998). Recently,it was shown that tumor lines established in vitro from ovarian carcinomas, as well as ovar-ian carcinoma cells harvested from malignant ascites, frequently overexpress the HER-2protein at their surface (Hellström et al., 2001). This evidence suggests that cells express-ing this protein have a selective growth advantage over HER-2-negative cells.

To further clarify the above-mentioned suggestion, ovarian SKOV3.A2 cells havebeen used for identifying the specific HER-2 chain for a better source of its epitopes(Castillega et al., 2001). It is known that these cells express high and stable levels of HER-2.In these cells, HER-2 is not autophosphorylated at Y1248 and other receptor tyrosinekinase (RTK) sites. As a consequence, the proliferation of these cells is less dependent onmitogenic signaling by tyrosine-phosphorylated HER-2 and is consequently less suscepti-ble to inhibition by geldanamycin. This drug is known to mediate its inhibitory effects onRTK by binding strongly to stress proteins such as heat shock protein or glucose-regulatedprotein, which forms complexes with HER-2. Such an inhibition occurs indirectly bydestabilizing stress protein–complexed kinases (Chavany et al., 1996). Geldanamycin doesnot inhibit HER-2 mRNA and protein synthesis in tumor cells, but by dissociating theHER-2-glucose–regulated protein complex, it reduces the protein half-life. Treatment of cellswith this drug increases the rate of ubiquitination of existing HER-2 protein with consequent

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faster proteasomal degradation. Also, HER-2 synthesized in the presence of geldanamycinis unstable and does not accumulate in tumor cells (Mimnaugh et al., 1996).

Like patients with breast carcinoma, those with ovarian cancer may benefit fromtreatment with Herceptin in combination with chemotheropeutic drugs. Herceptin treat-ment is effective, in both early and advanced stages of ovarian cancer (when the majorityof tumor cells express HER-2 protein), for eliminating the potentially more malignantHER-2-positive tumor cells. The effectiveness of Herceptin is based on the affinity of thismonoclonal antibody for the extracellular domain of HER-2, which is common as ovariancarcinomas progress. However, a tumor vaccine inducing antibody and/or T-cell immunityto HER-2 epitopes will ultimately provide the most effective means to prevent the emergenceof HER-2-positive cells.

Prostate Carcinoma

Prostate carcinoma is the most common cancer in men in the United States.Approximately 180,400 new cases were diagnosed in 2000, with 31,900 deaths as a resultof the disease. Prostate cancers typically begin with androgen-dependent lesions, but thecancer usually becomes androgen-independent when the disease is advanced. It has beenshown, for example, that androgen-independent LAPC-4 prostate cancer sublines expressa higher level of HER-2 than that expressed by their androgen-dependent counterparts(Carter et al., 2001).

Alterations in the signal transduction pathways mediated by HER-2 play an impor-tant role in the progression of prostate cancer. This protein is normally expressed at a verylow level in a few human secretory epithelial cells but is overexpressed in a number ofhuman cancers, including prostate carcinoma. Overexpression of HER-2 is an indicator ofpoor prognosis in prostate cancer patients.

Because of the deaths and considerable clinical complications that occur as a resultof prostate cancer, metastatic forms of the disease have been and remain an important tar-get for novel therapeutic interventions. Although standard hormonal therapy for metastaticprostate cancer has a high response rate (70–80%), hormone resistance ultimately devel-ops, which necessitates additional therapy. Novel therapeutic HER-2-specific immuno-toxins, such as scFv (FRPS)-ETA, have been developed and might be useful agents for thetreatment of prostate cancers with high levels of HER-2 protein. Another example is thedevelopment of bispecific antibodies (e.g., MDXH210) designed to direct the cytotoxiceffects of monocytes and macrophages to destroy tumor cells expressing HER-2. Theseconstructs are discussed elsewhere in this chapter.

Squamous Cell Carcinoma of Cervix

An inverse pattern of expression of HER-2 and epidermal growth factor receptor(EGFR) is found in normal tissues of the female genital tract and placenta (Wang et al.,1992). Although HER-2 is closely related in structure to EGFR, these two oncoproteinsmay play a different role in cell control. Ngan et al. (2001) have studied the expression ofHER-2 and EGFR at the protein level using immunohistochemistry, at the RNA level

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applying the ribonuclease protection assay, and at the DNA level using the Southern blotand hybridization method. Activation of proto-oncogenes can be determined by assessingthe level of RNA expression or DNA amplification. A large number of patients with squa-mous cell carcinoma of the cervix were recruited. EGFR showed a high percentage(74.2%) of overexpression with immunohistochemical staining and 35.4% of DNA ampli-fication in squamous cell carcinoma of the cervix. In contrast, HER-2 showed only 19 8%of overexpression with staining and 17.2% of DNA amplification. It is concluded that theabnormal expression of these two proteins has no prognostic significance on survival rates.

METHODS FOR DETECTING HER-2 STATUS

A number of methods are available for analyzing tumor HER-2 status. The selectionof the method depends on the target molecule to be detected. The target molecules are DNAmRNA, and protein (Fig. 12.2). HER-2 gene amplification can be detected by Southern blot(Press et al., 1994), slot blot (Naber et al., 1990), and dot blot assays (Descotes et al., 1993),fluorescence in situ hybridization (FISH) (Persons et al., 1997), in situ hybridization (ISH)on isolated nuclei or tissue sections (Smith et al., 1994), and polymerase chain reaction(Gramlich et al., 1994). Assays to determine mRNA overexpression include Northern blot(Slamon et al., 1989), Western blot (Press et al., 1994), slot blot (Naber et al., 1990) andISH (Naber et al., 1990). Methods to assess HER-2/neu protein product overexpression

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include Western blot analysis (Slamon et al., 1989), immunoassays (Dittadi et al., 1997),FISH (Lebeau et al., 2001), and immunohistochemistry (Pauletti et al., 2000).

Some of the above-mentioned methods have limitations. Southern blot analysisrequires a large amount of high-quality DNA, and the results are unreliable if the samplehas a low percentage of tumor cells (Ross and Fletcher, 1998). For these and other draw-backs, this method is precluded from becoming a routine clinical protocol to determineHER-2 status.

Polymerase chain reaction (PCR), on the other hand, has several advantages: it canbe used to analyze small numbers of tumor cells, DNA from formalin-fixed, paraffin-embedded tumor tissue can be used, and it can be automated and standardized. QuantitativePCR techniques are currently being assessed for their clinical application to HER-2 DNAtesting (Vona et al., 1999). However, presently the PCR technology is not optimally suitedfor routine, clinical application (see next section for details of quantitative analysis ofHER-2/neu expression).

It is apparent that many of the above-mentioned methods to analyze gene amplifica-tion and mRNA overexpression are beyond the scope of most pathology laboratories fortechnical reasons, and most of these assays require prospective collection of fresh tissueand thus are not applicable to archival tissue specimens.

Fluorescence in situ hybridization is more widely used than Southern blot and PCRtechniques and allows visualization of HER-2 DNA in individual cells using a specificfluorescence-labeled probe. Recently, Pauletti et al. (2000) have demonstrated HER-2/neugene amplification in infiltrating breast adenocarcinoma using FISH with a HER-2/neu-specific probe. Another elegant study has demonstrated three-color FISH images of breastcarcinoma cell nuclei from two primary tumors with HER-2 oncogene amplification(Järvinen and Liu, 2000). By performing dual-color FISH, the HER-2/neu gene status canbe quantified, and information about the ploidy status of chromosome 17 can also beobtained (Walch et al., 2001). The detection of gene amplification in individual tumor cellsis one of its major advantages. However, in the absence of standardization, results will dif-fer. For details about detecting HER-2/neu amplification with FISH, see Ross et al. (2001).This detection method in breast cancer is based on the Oncor INFORM HER-2/neu GeneSystem using a sequence biotinylated probe (Oncor, Gaithersburg, MD).

QUANTITATIVE ANALYSIS OF HER-2/NEU GENE EXPRESSION

It is well established that formalin-fixed, paraffin-embedded tissue is the most widelyavailable specimen for retrospective clinical studies. These specimens provide an invalu-able source for the elucidation of disease mechanisms and validation of differentiallyexpressed genes as therapeutic targets or prognostic indicators. However, the reliablequantitation of gene expression in formalin-fixed, paraffin-embedded tissues has seriouslimitations. Nucleic acids may be extracted from these specimens. Although this is a lesserproblem for DNA, RNA isolated from such tissues is of poor quality due to its degrada-tion before fixation is completed. Furthermore, formalin fixation crosslinks nucleic acidand proteins and covalently modifies RNA by adding monomethylol groups to the bases.This modification becomes a problem in subsequent RNA extraction, reverse transcription,and quantitative analysis.

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To cope with the above-mentioned problem, quantitative gene expression analysis canbe accomplished in combination with laser-assisted microdissection in formalin-fixed,paraffin-embedded tissues. Using an optimized RNA microscale extraction procedure inconjunction with real-time quantitative reverse transcriptase-polymerase chain reaction(QRT-PCR) based on fluorogenic TaqMan methodology, Specht et al. (2001) have ana-lyzed the expression of a panel of cancer-relevant genes, including HER-2/neu. They used54 microdissected nonneoplastic and neoplastic archival samples from patients withBarrett’s esophageal adenocarcinoma for analyzing HER-2/neu expression with the QRT-PCRand compared it with that obtained in parallel with fluorescence in situ hybridization andimmunohistochemistry. The results of these three methods matched one another.

The QRT-PCR has been used for comparing quantitative mRNA expression ofHER-2/neu with DNA and oncoprotein levels in the metaplasia-dysplasia-adenomacarci-noma sequence of Barrett’s adenocarcinoma (Walch et al., 2001). Tissue sections from thesame area can be used for QRT-PCR of laser-microdissected tumor cells and for immuno-histochemistry. The method has been successful in quantifying HER-2/neu gene expres-sion in small microdissected tissue samples from archival material as well as in premalignantlesions (Specht et al., 2001; Walch et al., 2001). Only a locus-specific HER-2/neu geneamplification is associated with strong mRNA overexpression and strong plasma mem-branous immunostaining in Barrett’s adenocarcinoma. Quantitative PCR techniques arecurrently being assessed for their clinical application to HER-2 DNA testing (Vona et al.,1999). However, at present PCR technology is not optimally suited for routine, clinicalapplication.

The QRT-PCR is a sensitive, accurate, and highly reproducible method for studyinggene expression. The method is based on the 5' nuclease activity of Taq DNA polymeraseand involves cleavage of a specific fluorogenic hybridization probe that is flanked by PCRprimers spanning an amplicon range of 60–150bp (Gibson et al., 1996). It is capable ofdetecting PCR products as they accumulate during amplification. The reactions are char-acterized by the point during cycling when PCR amplification is still in the exponentialphase, allowing precise quantitation of RNA over a wide dynamic range. Because of thesmall target size, this approach is suitable for quantitative determination of gene transcriptlevels, even in tissue extracts containing partially fragmented RNA. Because only smallamounts of RNA are required, this technique is applicable to small clinical biopsies andmicrodissected cell clusters from frozen or formalin-fixed, paraffin-embedded tissue sec-tions (Specht et al., 2001). Laser-assisted microdissection is indispensable for the selectiveanalysis of stroma-free tumor cell populations, circumventing the problem of tissue het-erogeneity as well as providing the possibility of assigning characteristic gene expressionpatterns to particular histological phenotypes (Simone et al., 1998). This methodologyopens new avenues for the investigation and clinical validation of gene expression changesin archival tissue specimens.

DETECTION OF HER-2 ONCOPROTEIN

Three main techniques have been used for detecting HER-2 oncoprotein overexpression:Western blot analysis, enzyme-linked immunosorption assay (ELISA), and immunohisto-chemistry. Western blot analysis is limited to basic research rather than routine clinical

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analysis of HER-2 status because it requires fresh tumor homogenates. The use of tumorhomogenates results in large variations, depending on the amount of stromal and othernontumor cells present in the homogenate, causing a potential dilution effect (Ross andFletcher, 1998).

Enzyme-linked immunosorption assay can be used to measure HER-2 protein intissue homogenates or in serum. It is a relatively simple technique and is well suited toautomation (van de Vijver, 2001). However, when tumor cytosolic fractions are used, his-tological information is lost and invites the undesirable dilution effect. Therefore, theELISA assay is not used routinely to determine HER-2 status.

On the other hand, immunohistochemistry facilitates the identification of HER-2 proteinoverexpression in individual tumor cells and has become the most common approach fordetermining HER-2 status. Numerous reasons for its advantages are detailed throughoutthis book. As is true in many other techniques, routine immunohistochemistry has thedisadvantage of being nonstandardized. One way to significantly minimize interlaboratoryvariability and achieve a degree of standardization is to use a single commercial test kitsuch as the HercepTest (Dako) discussed below. Another limitation of immunohistochem-istry is that it relies on subjective interpretation of staining results. Many variables affectstaining results, which are discussed in Chapter 5 and elsewhere in this book.

Table 12.1 indicates one variable, i.e., antibodies, that affects HER-2 protein positiv-ity. This table also indicates the role of different antibodies in determining HER-2 positiv-ity in breast tumors, using immunohistochemistry.

In conclusion, immunohistochemistry and FISH are recommended for HER-2/neuanalysis in both routine clinical practice and in clinical research studies, using sections offormalin-fixed, paraffin-embedded tissues. Although these two methods show a high levelof correlation with each other in the evaluation of HER-2/neu status of breast cancer,immunohistochemistry is preferred for routine use. The FISH procedure requires moretime and expense than immunohistochemistry. In addition, the FISH slides must be storedat –20°C or lower and are prone to quenching of the fluorescent signal with time. Moreimportant, immunohistochemistry provides information on immunostaining as well as oncell morphology of the same section. However, according to Pauletti et al. (2000), FISHprovides superior prognostic information in segregating high-risk from low-risk breastcancers than that obtained with immunohistochemistry. Antibodies used in this procedure

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are discussed later. Excellent studies comparing these two methods have been carried outby Jacobs et al. (1999) and Lebeau et al. (2001).

Controversy continues over whether HER-2/neu gene amplification or gene productp185 overexpression is the best predictor of therapy response and clinical outcome. Becauseof this uncertainty, it is apparent that further clinicopathological studies are required todemonstrate whether HER-2/neu amplification or protein overexpression, or a combinationof the two methods, has prognostic value in breast cancer. The most widely applied tech-niques to detect the amplification of this gene and the overexpression of its protein productare FISH and immunohistochemistry, respectively. The ideal approach is to correlate quan-titative FISH with immunohistochemistry using antigen retrieval and computer-assistedimage analysis. The results are further improved when immunohistochemical studies areassessed as the difference between tumor cells and nonneoplastic breast tissue. In otherwords, immunostaining intensity of nonneoplastic breast tissue is subtracted from the neo-plastic staining, correcting for cytoplasmic background staining. Only distinct membranousimmunostaining of HER-2 has prognostic value in breast cancer, while occasional cyto-plasmic staining is without such relevance. Both techniques allow the study of smallamounts of formalin-fixed, paraffin-embedded tissue and the interpretation of the findingson a cell-by-cell basis. Using this approach, Lehr et al. (2001) have convincingly demon-strated a high degree of concordance between the two techniques in breast cancer.

BISPECIFIC ANTIBODIES

Bispecific antibodies are chemically or genetically linked antibodies with two het-erologous antigenic specificities. Potentially antineoplastic bispecific antibodies can beprepared by combining specificities for a tumor antigen and cytotoxic trigger molecules onimmunoeffector cells. Such studies were developed in order to target cytotoxic immuno-effector cells to tumors to facilitate antibody-dependent cell toxicity (see also Chapter 2).Essentially, bispecific antibodies (e.g., MDX-H210) are novel antibody constructsdesigned to direct the cytotoxic effects of monocytes and macrophages to the destructionof tumor cells expressing HER-2.

Recently, Sen et al. (2001) have described methods for generating highly effectiveHER-2-specific cytotoxic T cells by arming activated T cells with anti-CD3 X anti-HER-2bispecific antibody. In this method OKT3 and 9184 anti-HER-2 monoclonal antibodieswere conjugated and used to arm T cells that were subsequently tested for binding,cytotoxicity, and cytokine secretion assays. Armed T cells aggregate and selectively killHER-2-positive breast cancer cells (MCF-7).

Such cytotoxic T cells can be produced by arming activated T cells with nanogramquantities of OKT3/9184. Advantages of this technique are that arming activated T cellswith low doses of the bispecific antibody obviates the need for administering large amountsof the antibody required for infusional therapy, and cytotoxicity is augmented by combin-ing antibody targeting and T cell–mediated killing. Also, binding of effector cells at thetumor site by armed activated T cells may augment tumoricidal activity, as well as increaselocal cytokine secretion leading to recruitment of other immune effectors. This approach isunique; in future clinical trials, billions of activated T cells could be armed with milligramsof the bispecific antibody, thus becoming HER-2-specific cytotoxic T lymphocytes.

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A recombinant fusion protein [scFv(FRP5)-ETA] consisting of an HER-2-specificsingle-chain antibody and the Pseudomonas exotoxin A has been developed for evaluatingits cytotoxicity on HER-2 expressing an established human prostate cancer cell line(LNCaP) (Wang et al., 2001). This cell line expresses high levels of HER-2 protein.Exposure of these cells to scFv(FRP5)-ETA causes significant cell death and reduces thelevel of prostate-specific antigen. Based on this evidence, scFv(FRPs)-ETA might be con-sidered a useful agent for treating human prostate cancer cells with high levels of HER-2expression.

Bispecific Antibody MDX-H210

MDX-H210 is a partially humanized Fab' X Fab' bispecific antibody constructed bychemical conjugation of the F(ab') fragments of the murine monoclonal antibody 520C9(anti-HER-2) having specificity for the cell surface region of the HER-2/neu gene productand H22, a humanized monoclonal antibody that binds to the human immunoglobulinreceptor (CD64). is a 72-kDa protein that is one of three receptors expressedon the plasma membrane of monocytes, macrophages, and or G-CSF stimulatedPMNs (Lewis et al., 2001). Monclonal antibody H22 binds to a site outside the ligandbinding domain for IgG yet effectively triggers cellular responses in thepresence of high concentrations of human (serum) IgG. In other words, cytotoxicity is notblocked by IgG or serum.

Lewis et al. (2001) have studied the combination of with MDX-H210 based onthe hypothesis that would activate and up-regulate the expression of onneutrophils, monocytes, and macrophages. This approach facilitates an interactionbetween expressing effector cells and tumor cells expressing HER-2, thus enhanc-ing destruction of tumor cells. Humanized MDX-H210 has dual specificity and immuno-logical activity in vitro; that is, it causes lysis of HER-2-expressing cell lines mediated bymonocytes and and G-CSF-activated PMNs.

Recently, a phase 1 pilot trial of the MDX-H210 in patients whose prostate canceroverexpressed HER-2 was carried out by Schwaab et al. (2001). They undertook this trialto direct the cytotoxic effects of MDX-H210 on monocytes and macrophages through

to destroy prostate tumor cells expressing HER-2. This agent was well tolerated atdoses that appeared to be immunologically and clinically active. At these dosesbiological activity was demonstrated and characterized by binding of the antibody to cir-culating monocytes, release of monocyte-derived cytokines, a decrease in circulatingHER-2 protein, and short-term stabilization of prostate-specific antigen levels. However,patients receiving an intravenous infusion of MDX-H210 did show malaise, fever, chills,and myalgias. A larger phase 2 study of this antibody combined with cytokine (GM-CSF)is underway by these workers.

In another recent phase 1 trial, MDX-H210 was combined with for increasingthe expression of as well as activating the monocyte/macrophage immunoeffectorcells in patients with advanced cancer that overexpressed HER-2 (Lewis et al., 2001).Among the 23 patients, 19 had breast cancer, 3 had prostate cancer, and 1 had lung cancer.This study indicated the absence of a positive relationship between maximum plasma con-centration of MDX-H210 or peak plasma cytokine concentrations and clinical toxicity.

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VACCINES

Recently, there has been renewed interest in developing vaccines for use in cancer treat-ment. The main factors giving impetus to this therapeutic approach include a better under-standing of the immune system, the identification of several T cell–specific tumorantigens, more effective adjuvants, and the ability to construct more immunogenic mole-cules using recombinant DNA techniques (Murray et al., 2000). Current vaccine strategiesfor the treatment of solid tumors tend to focus on the cellular arm of the immune response.

The overexpression of HER-2 protein in cancer cells makes it an ideal target for vac-cines and other targeting strategies. Vaccines optimized to induce maximum T cell immu-nity to HER-2 may lead to potent in vivo antitumor immunity. HER-2 protein has beenevaluated as a potential target for the development of cancer vaccines because preexistentT cell and antibody responses to HER-2 have been described in breast cancer patients(Disis and Cheever, 1996). In other words, breast cancer patients have preexisting immu-nity to the HER-2 receptor in the form of elevated antibody titers and T cell immunity.Elevated anti-HER-2 T cell responses have been demonstrated in breast and ovarian can-cer patients following immunization with peptides derived from the HER-2 protein (Disiset al., 1999). However, whether peptide-specific T cell responses can be translated toantitumor immunity has yet to be established.

A recent study has utilized an in vivo murine tumor model expressing human HER-2for evaluating potential HER-2 vaccines consisting of either full-length or variable subunitsof HER-2 delivered in either protein or plasmid DNA form (discussed later) (Foy et al.,2001). The mechanism of protection elicited by plasmid DNA vaccination appears to beexclusively CD4-dependent and not CD8- or antibody-dependent, whereas the protectionobserved with intracellular domain protein vaccination requires both CD4 and CD8 T cells.However, the exact mechanism(s) responsible for immunity to DNA has not been elucidated.

Another recent study supports the use of the dendritic cell-based vaccine as a thera-peutic strategy to target both CD4 and CD8 T cells to HER-2 (Chen et al., 2001). To min-imize the possibility of deleterious effects, the transforming activity of the HER-2molecule can be inactivated by a single amino acid substitution (lysine to alanine), unlikeother studies in which the entire intracellular domain was removed.

Genetic Immunization

Genetic immunization, also known as DNA or polynucleotide immunization, is anovel strategy for vaccine development in the host. In this technique plasmid DNA encod-ing either individual or a collection of antigens is directly administered to a host. As aresult, the delivered foreign gene is expressed by the host, which in turn leads to the induc-tion of a specific immune response against the in vivo produced antigen. DNA immuniza-tion has been shown to induce protective immune responses in several infectious diseaseand cancer experimental model systems (Cohen et al., 1998). These vaccines have alsoentered the clinical trials both as protective and as therapeutic agents.

Unlike peptide vaccines, which must be specific for each individual’s MHC, expres-sion of plasmid-encoded tumor antigens within the host antigen-presenting cells followingvaccination results in the presentation of multiple tumor–associated epitopes in the context

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of MHC class I and/or class II molecules. Direct intramuscular injection with DNA plasmidexpressing HER-2/neu tends to induce antigen-specific cellular and humoral responses inmice (Wei et al., 1999).

Chen et al. (1998) have shown that plasmid DNA encoding a truncated rat erbB-2/neuthat lacked the intracellular domain could induce protective immunity against erbB-2/neu–expressing mammary tumors as effectively as plasmid encoding the full-lengtherbB-2/neu oncogene. However, more recent clinical trials demonstrated that CD4 T cellsrecognizing peptides from both extracellular and intracellular domains of human erbB-2/neuprotein can be induced by peptide vaccination. Vaccination with full-length DNA throughex vivo targeting of the dendritic cells has the advantage of presenting the complete repertoireof erbB-2/neu epitopes in association with MHC class I and class II molecules, maximizingthe number of peptide epitopes available for T cell recognition (Chen et al., 2001).

DNA vaccination requires host dendritic cells for priming the T cell response, eitherthrough direct transfection or antigen transfer from transfected nonhematopoietic cells.Because dendritic cells are functionally impaired in cancer patients, antigen processingand presentation following genetic immunization may be inefficient. However, this prob-lem can be overcome by differentiating ex vivo dendritic cells from precursors present inthe peripheral blood. The ex vivo cultured dendritic cells are fully functional and can beused as cellular vectors for vaccines (Dhodapkar et al., 1999).

IMMUNOHISTOCHEMISTRY

Immunohistochemistry can be used, with or without antigen retrieval, for localizingHER-2/neu gene protein (p185). In the absence of antigen retrieval, immunoelectronmicroscopy can also be employed for detecting this protein in different cell types at theultrastructural level. Recently, this method was used for detecting p185 in laryngeal squa-mous cell carcinoma with the electron microscope (Grzanka et al., 2000). The use of rab-bit antihuman HER-2/neu oncoprotein (Dako) (diluted 1:100) demonstrated antigenicitynot only on the plasma membrane, but also in the rough endoplasmic reticulum. It isknown that the protein moiety of this glycoprotein is synthesized in the rough endoplasmicreticulum, and glycosylation subsequently occurs in the Golgi complex.

Most studies have been carried out using immunohistochemistry with the light micro-scope. HER-2/neu gene protein (p185) expression can be investigated with the mousemonoclonal antibody (mAbl) (Triton Diagnostic, Alameda, CA), an IgGl immunoglobulinthat recognizes the external domain of this protein. This antibody has been used at a dilu-tion of 1:200 for targeting this gene product in primary breast cancer tissue, without anti-gen retrieval pretreatment (Fig. 12.3) (Horiguchi et al., 1994). Alternatively, HER-2/neugene product expression can be localized in breast cancer tissue using the rabbit polyclonalantiserum (R60), which targets the intracellular domain of p185 (Pauletti et al., 2000).Tissue sections do not require an antigen retrieval treatment.

Another polyclonal antibody, Anti-c-erb-B2, can be used in conjunction with antigenretrieval in a microwave oven according to the standard procedure presented in thisvolume. This protocol has been used for targeting c-erbB2 (HER-2/neu) gene protein inthe ovarian surface epithelial tumors (Anreder et al., 1999). Polyclonal antibody pAbl(Triton Biosciences) can also be used for assessing immunostaining (both the intensity and

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extent) of the (HER-2/neu) gene product in breast cancer tissue (Kay et al., 1994). Fordetecting this gene product in bladder, lung, and renal tumors, monoclonal antibody NCL-CB11 has been used (Kay et al., 1994). This antibody recognizes the internal domain(cytoplasmic staining) of the HER-2/neu gene product. The expression of this oncoproteinhas also been studied in mammary Paget’s disease using three different antibodies (Edorhet al., 1995). Antibodies NCL-CB11 and NCL-CBE1 were used for recognizing internaland external domains, respectively, of this protein.

As indicated above, a number of different monoclonal and polyclonal antibodies havebeen used for determining the HER-2/neu status in breast cancer. A wide range ofHER-2/neu overexpression rates has been reported in different studies, varying from 9–60%.This range resulted from using a wide variety of antibodies and/or variations in preparationtechniques, scoring criteria, and stage of cancer. The HercepTest containing rabbit-polyclonal antibody A0485 (Dako) is superior to other antibodies. Recently, this antibodywas compared with five monclonal antibodies (9G6, 3B5, CB11, TAB 250, GSF-HER 2) andone polyclonal antibody (A8010), using formalin-fixed, paraffin-embedded archival invasivebreast cancer tissues (Lebeau et al., 2001). This study has confirmed the superiority of A0485over other antibodies. Another study also indicates the superiority of A0485 over polyclonalantibody available from Oncor, Inc. (Gaithersburg, MD) (Maia, 1999). In conclusion, thetype of antibody used will affect the results of HER-2/neu protein immunohistochemistry.Figure 12.3 shows the immunostaining of HER-2 oncoprotein in invasive breast carcinoma,using monoclonal anti-c-erbB-2 antibody. Table 12.2 shows immunohistochemical localiza-tion of this protein in different carcinomas in various tissue and organ types.

HERCEPTIN (TRASTUZUMAB)

A large body of evidence in the literature supports the idea that antibodies directedagainst HER-2 can inhibit the growth of HER-2–expressing tumors through several mech-anisms, including antibody-dependent, cell toxicity–mediated inhibition of HER-2 signaltransduction.

A number of murine monoclonal antibodies against the extracellular domain of theHER-2 protein have been found to inhibit the proliferation of human cancer cells that over-express HER-2 both in vitro and in vivo (Hudziak et al., 1989; Shepard et al., 1991). Tominimize immunogenicity, the antigen binding region of one of the more effective anti-bodies (Herceptin, Ab4D5) was fused to the framework region of human IgG and testedagainst breast cancer cells that overexpress HER-2 in vivo and in vitro (Carter et al., 1992;Pietras et al., 1994). It is well established that Herceptin is superior to other antibodies(e.g., polyconal antibody, Oncor, Inc.) and is called recombinant humanized anti-HER-2antibody. Although Herceptin inhibits tumor growth when used alone, it has synergisticeffects when used in combination with chemotherapy.

Phase 1 clinical trials demonstrated that Herceptin is safe and confined to the tumor.However, phase 2 trials indicated that the efficacy of the antibody is superior when givenwith chemotherapy (Pegram et al., 1998). Phase 3 trials have indicated that Herceptin,when added to conventional chemotherapy, can benefit patients with metastatic breastcancer that overexpresses HER-2, prolonging relapse and overall survival (Slamon et al.,2001). Similarly, extensive studies by Menden et al. (2001) support the weekly Docetaxel

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and Herceptin combination therapy for women with HER-2 overexpression metastaticbreast cancer. This combined treatment is thought to be well tolerated with significant anti-tumor activity. However, a most troubling adverse effect of this antibody is cardiac dys-function, especially when given concurrently with an anthracycline. The mechanism of thecardiotoxicity of Herceptin is not known. Therefore, great caution is required in using thisantibody. Additional clinical trials are needed to further evaluate and confirm the usefulnessof Herceptin when used in combination with chemotherapy.

HERCEPTEST

The immunohistochemistry of estrogen receptors and progesterone receptors pro-vides valuable information, aiding in the selection of breast cancer patients for endocrinetreatment. However, consensus on the effectiveness of this approach is lacking.Immunostaining of HER-2/neu gene product (HER-2), on the other hand, is an effectiveprognostic indicator in patients with breast cancer. In 1998 the U.S. Food and DrugAdministration (FDA) and the Health Protection Branch of Canada approved HER-2 usefor the HercepTest for immunohistochemical detection of HER-2 overexpression in breasttumors of patients who are being considered for Herceptin (trastuzumab) therapy. Unlikemany chemotherapeutic agents that destroy any population of dividing cells, Herceptintargets a specific molecular abnormality which is absent on nonneoplastic cells.

Dako’s immunohistochemical assay is the only FDA-approved protocol for detectingHER-2 oncoprotein overexpression. Dako’s kit is used for analyzing this oncoprotein insections of paraffin-embedded breast tumors using immunohistochemistry or FISH. Thesetwo assays determine the patient’s HER-2/neu status. Immunostaining of the HER-2/neugene product and the detection of oncogene amplification demonstrate good correlation.Gene amplification detected by FISH is 92% concordant with immunohistochemicaldetection of overexpression of the oncoprotein (Persons et al., 1997); however, the percentagemay be higher for HER 3+ cases.

Although the two methods mentioned above are viable options, they should not beapplied independently of each other. A tested, standardized, and well-controlled immuno-histochemical assay should serve as the first test. It is simple to carry out and quite reliablefor the two extreme ends of the staining spectrum (9 and 3+) (Hendricks, 2000). The 2+cases (weak staining in at least 10% of neoplastic cells) can in turn be subjected to FISHanalysis to confirm the presence of an altered HER-2/neu gene.

The results of the HercepTest are interpreted in a semiquantitative manner, withscores ranging from 0–3+, reflecting the intensity of the staining reaction (Table 12.3).Thus, Herceptin therapy is based on the difference between faint and weak staining.Contrary to some other immunohistochemical assays, which are based on the percentageof positive cells, the HercepTest immunohistochemical assay is based on both the intensityand extent of staining. The subjective nature of judging stain intensity invites bias evenwhen positive controls are included in the study (Hendricks, 2000).

For the HercepTest to be valid, it must be performed exactly according to the manu-facturer’s directions. The tissue must be fixed in 10% neutral buffered formalin or Bouin’ssolution. Sections of paraffin-embedded tissues should be cut thick and processedin a standard tissue processor. Epitope retrieval should be carried out in a water bathinstead of in a microwave oven. Water is used because it provides more uniform and

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consistent heating than do other heating methods. Any variation in the manufacturer’sinstructions can lead to variability in results.

Indeed, interobserver and interlaboratory variability of the interpretation of immunohis-tochemical staining using the HercepTest is not uncommon for HER 1 + cases, because inthese cases it is not clinically relevant. In other words, scores of 0 and 1 + are considered neg-ative and indicate probable nonresponsiveness to Herceptin. The variability is applicable forHER 2+ and 3+ cases. The scoring systems and cutoffs used in different studies may vary.Therefore, there is a need for optimizing the staining evaluation system. This variability canbe significantly minimized by quantifying HER-2 expression by image analysis (Press et al.,1993). In a recent elegant study, Hatanaka et al. (2001) have quantified the levels of HER-2protein expression in breast cancer using an image analyzing system and applied this systemfor optimizing the interpretation of the HercepTest. By converting the quantitatively extracteddata into a scoring system based on the criteria, the outcome demonstrates a strong concor-dance with the scoring data obtained from immunostaining. Figure 12.4 (Plate 6) shows theimage analysis of breast carcinoma immunostained with the HercepTest. In this system,brown and blue signals are extracted and replaced with artificial green images.

A problem with the HercepTest arises when it is used at high altitudes. One of therequirements of this test is that the slides must be heated in Epitope Retrieval Solution(Dako) in a water bath at 95°C for 40 min. While this temperature can be easily obtained atsea level, it is difficult to achieve at altitudes above 5,000 feet. At these altitudes, water boilsrapidly below 95°C as a result of air pressure on the boiling point. The use of boiling waterdoes not conform to the manufacturer’s directions. Various solutions to this problem includeextending the duration of heating to 50 min at 90°C or placing a metal lid over the waterbath. Any adjustment in the heating step should be assessed using the 1 + control cell lineincluded in the HercepTest kit. Ruegg and Lupfer (2001) have summarized various solutionsto this problem.

Controls and Scoring System

For the negative control, primary antibody is replaced with an irrelevant, isotype-matched antibody. This step controls nonspecific binding of the secondary antibody. The

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positive control slides consist of sections of cell blocks of the three breast cancer cell linesSKBR3, MDA-MB-175, and MDA-MB-231, which express 2.4 million, 92,000, and22,000 HER-2 receptor molecules, respectively, by Scatchard analysis (Koeppen et al.,2001). These receptor numbers correspond to immunohistochemical HER-2 scores of 3, 1,and 0. These scores are defined by a lack of staining or membranous staining in less than10% of the cells (0), incomplete membranous staining in more than 10% of the cells(score 1), and complete membranous staining of strong intensity in more than 10% of thecells (score 3). Scores of 2 or higher indicate HER-2 overexpression. A score of 2 or higheris generally considered to be staining of 20–35% of the cells. Only membranous stainingshould be considered as positive.

The incidence of HER-2 overexpression varies considerably in different tumors.Table 12.4 shows an average score of HER-2 overexpression in representative human solidtumors commonly evaluated in clinical practice. The specimens in this study consisted ofresections of primary tumors. Locally advanced and metastatic lesions of the same tumortypes may show significantly different incidences of HER-2 overexpression. Infiltratingductal carcinoma is one of the few tumor categories for which the incidence of HER-2overexpression is consistent in most studies. These data were obtained using a single,standardized immunohistochemical method (Herceptin).

IMMUNOSTAINING OF HER-2 PROTEIN USING HERCEPTEST

Breast tumor tissues are fixed with formalin for 24 hr and embedded in paraffin, andsections are deparaffinized according to standard procedures. They are heated in 0.1 mMsodium citrate buffer (pH 6.0) in a water bath for 40 min at 95°C. After cooling for 20 min,the sections are treated with 0.3% hydrogen peroxide containing 15 mM sodium azide for

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10 min to block endogenous peroxidase activity. They are thoroughly rinsed in a washingbuffer (50 mM Tris-HCI buffered saline [pH 7.6] containing a detergent), incubated withanti-HER-2/neu antibody for 30 min, and washed three times with the washing buffer.

The antibody, bound to HER-2 protein, is detected by incubation (for 30 min) with thedextran polymer reagent conjugated with peroxidase and secondary antibody. Followingwashing with the washing buffer, color development is achieved with DAB for 10 min inan automatic stainer. As controls, slides containing three cell lines showing different HER-2protein expression are included in the staining protocol. This step is used to confirm vali-dation of the staining results.

Semiquantitative analysis is carried out based on a score of 0 (no staining or membranestaining on less than 10% of tumor cells), 1+ (a faint perceptible staining), 2+ (a weak tomoderate staining of the entire membrane), and 3+ (a strong staining of the entire mem-brane). Scores of 1+ to 3+ are also essential to be positive for more than 10% of tumorcells. Scores of 0 or 1+ are negative for HER-2 protein overexpression, while scores of2+ and 3+ are weak positive and strong positive, respectively. Only membrane staining isevaluated.

Quantitative analysis of the HER-2/neu expression is calculated using a color videocamera with image processing software. The three images for analysis are picked up fromtumor lesions that exhibit predominant and typical features microscopically in each case(Hatanaka et al., 2001). The quantitative labeling index for assessment is calculated by theratio of brown membranous staining area stained with DAB to round blue areas stainedwith hematoxylin in consideration of tumor cell density in selected image. The extractionof a brown or blue signal is carried out with specific protocols based on RGB color param-eter, selected automatically in accordance with the protocols, and highlighted as artificialgreen images (Fig. 12.4/Plate 6).

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Index

ABC method: see Avidin-biotin methodAgNOR: see Nuclear organizer-associated

regionAlkaline phosphatase anti-alkaline phosphatase,

89, 99Aluminum chloride, role in antigen

retrieval, 76Amyloid fibril protein, 123Androgen receptor, 83, 87, 107, 143, 144, 146Angiogenesis, 20–25

in breast cancer, 22, 23in prostatic cancer, 22in vasculogenic mimicry, 21

Antibody, dilution of, 80, 82penetration of, 79specificity of, 3

Antibody-antigen interaction, mechanism of,72, 73, 79, 82, 83

Anticancer monoclonal antibodies, 47, 48Anticancer vaccines: see VaccinesAnti-cytokeratin 8 antibody, 118Antigenicity, preservation of, 72Antigen retrieval, 1, 2

in archival tissues, 173, 174in frozen brain tissue, 198, 199general method of, 169–172with heat, 124

advantages of, 124mechanisms for, 117, 130, 131

Antigen retrieval fluids, 75–77, 143molarity of, 74pH of, 74, 78

Antigens, denaturation of, 72, 73Antivimentin antibody, 118APAAP: see Alkaline phosphatase anti-alkaline

phosphatase

Apoptotic cells, immunodetection of, 201, 202Astrocytic tumor, HER-2

location in, 285Autoclaving, 145, 146

epitope retrieval with, 117method of, 128, 129, 145, 148

Autostainers, 138, 153Avidin-biotin method, 89, 90, 92, 98, 101

Background staining, 133, 142, 146–148bcl-2, 136, 153, 154Biotin, endogenous, 98–101

role in background staining, 97Biphasic mesothelioma, 25Bispecific antibodies, 44–46, 293, 294

development of, 45, 46Bladder tumor, 255

HER-2 location in, 285Blocking solution, 146Boric acid, as antigen retrieval fluid, 76, 88, 135Bouin’s fixative, 53, 59, 71, 96BrdU: see Bromodeoxyuridine indexBreast carcinoma, 75, 107, 118, 123, 129, 135,

138, 152, 210, 241, 263, 266, 269, 270,282, 283, 301

Bromodeoxyuridine index, 39, 40

Calcineurin, 145, 153Calcium ions, 117, 119, 147

modification of protein with, 120role in antigen masking, 120–122

effect of pH, 122role of monoclonal antibodies, 122

Carbohydrate antigens, 205–209retrieval with enzyme digestion, 208, 209retrieval with heat, 208

351

352 Index

CARD method: see Catalyzed reporterdeposition

Carnoy’s solution, 58, 59Catalyzed reporter deposition, 90, 92CD, definition, 44Cell proliferation index: see Labeling indexCell smears, immunostaining of, 182Chelating agents, epitope unmasking with, 120,

121Colon carcinoma, 85, 132Confocal scanning electron microscopy,

microwave heat-assisted, 230Conventional oven, heating in, 175Correlative microscopy, microwave

heat-assisted, 230, 231Cross-reactivity, 144; see also Monoclonal

antibodiesCryopreservation, 65Cryosections, immunostaining of, 200, 201,

245Cyclin D1 immunostaining, 184, 185Cytokeratin, 147, 154, 162, 176

DAB as chromogen, 72, 105, 106DAB-imidazol-copper sulfate, as chromogen,

98DCC: see Dextran-coated charcoal assayDetergents, 148–151

epitope retrieval with, 117, 118Dextran-coated charcoal assay, 5, 276Diagnostic pathology, 1Digestive enzymes, 8DMP-30 accelerator, 160, 161Double immunofluorescence staining, 186, 187Double indirect immunofluorescence staining,

187–189Double immunostaining, 182–184Ductal carcinoma, 85, 184, 254, 271, 279

EDTA as antigen retrieval fluid, 76, 101,121–124, 153

EDTA-NaOH as antigen retrieval fluid, 78Egg white, role in blocking endogenous biotin,

100Electron microscopy, 155–168ELISA: see Enzyme-linked immunosorbent

assayEndogenous calcium effect on antigen retrieval,

120–122Endogenous peroxidase, 1, 146

EnVision staining system, 99, 138–140procedure of, 138–140

Enzyme digestion for epitope retrieval, 117,118, 151–153

method of, 152Enzyme-linked immunosorbent assay

(ELISA), microwave heat-assisted,228–229

Epidermal growth factor, 22Epitope retrieval: see Antigen retrievalEpitopes, 1, 2, 73EPOS staining system, 138, 139Estrogen receptor alpha 265–267Estrogen receptor beta 267–268Estrogen receptor gamma 268Estrogen receptors, 74, 76–78, 85, 103, 106,

130, 143, 153, 261–279antibodies for, 261–279distribution in carcinomas, 264distribution in tissues, 268, 269immunohistochemistry of, 273–275immunostaining in prostate tissue,

277, 278role in breast cancer, 269, 270

semiquantitative assessment of, 276, 277Ewing’s sarcoma, HER-2 location in,

285, 286

False-negative staining, 60, 76, 80, 84, 102,104, 105, 149

False-positive staining, 10, 74, 78, 80, 97–99,104, 105

Fibronectin detection with monoclonalantibodies, 114, 197

Fine needle aspirates, 278Fixation, advantages and limitations of, 53Flow cytometry, detection of antigens with,

225–228Fluorescence in situ hybridization, microwave

heat-assisted, 222gastrointestinal neoplasia, 222, 223

Formaldehyde, 53–60effect of prolonged fixation with, 58, 59fixation with, 57mechanism of fixation with, 54–56nature of, 54

Formalin, 60overfixation with, 60

Formalin substitute fixatives, 59, 60Frozen sections, 136–139

Index 353

Gastric cancer, 123Genetic instability, 12, 13Glass slides, silanting of, 68, 69Gleason grading system, 111–113Glutaraldehyde, 56

effect of heating on, 61penetration of, 62properties of, 61

Glycerin, as antigen retrieval fluid, 77, 78Glycine-HCl buffer, 75Glycine-HCl buffer-EDTA, 75

as a denaturant, 150, 151

Heating methods, 73, 126–130autoclave, 128hot plate, 129, 130, 175–177hot water bath, 130microwave, 126, 127pressure cooker, 127steam, 128, 129water bath for electron microscopy, 178

for light microscopy, 178–180for free-floating sections, 180

Hepatocellular carcinoma, 99HER-2/neu oncogene, 22, 281–303

amplification of, 282quantitation analysis of, 290, 291

HER-2 oncoprotein, 22, 100, 283antibodies for, 292

bispecific antibody MDX-H, 210detection methods for, 289, 291–293distribution in carcinomas, 285, 297immunohistochemistry of, 296–298, 302, 303overexpression of, 284–289

in human solid tumors, 302scoring system for, 300–302simultaneous expression of p53, 284

HercepTest, 299, 300semiquantitative analysis of, 303

Herceptin, 11, 48, 298, 299HIER buffer, 77, 130Hirschsprung’s disease, 102Hot or cold spots, 77, 102, 103, 141, 143Hot plate heating

epitope retrieval with, 117method of, 129, 130

Image analyzer (SAMBA 4000), 106Immunofluorescence staining, 185Immunogold–silver staining, 167, 168

Immunogold staining, 167ImmunoMax method, 92In situ hybridization, 213–223

of DNA, 218, 219microwave heating with, 217, 218of mRNA in plant tissues, 221nonradioactive probes, 215–217radioactive probes, 215of RNA, 219

Interleukin (cytokine), 125Intestinal mucosa, 137Intrahepatic cholangiocellular carcinoma,

HER-2 location in, 286Intrasalivary lymphoid, 170

Ki-67 antigen, 10, 11, 20, 75, 79, 85–87, 106,122, 123, 132, 133, 139, 152, 154,233–241, 255

antibodies for, 237–239distribution in carcinomas, 239immunohistochemistry of, 235–237

limitations of, 237retrieval with autoclaving, 240, 241retrieval with microwave heating, 240

Kidney tissue, 7Kryofix fixative, 41, 60

Labeling index, 39BrdU, 39, 40MIB-1 antibody, 39, 40

Lab-SA kit, 99, 100Laryngeal squamous cell carcinoma, HER-2

location in, 286Ligand-binding assays, 275, 276Longer heating durations, 135Lung adenocarcinoma, 258

Malignant melanoma, 20, 134MIB-1 monoclonal antibody, 39–41, 79, 86,

152, 170, 209, 235–239, 240cross-reactivity of, 41dilution of, 80prognostic value of, 40

MICA method, 101Microarray technology, 18–20, 47

bladder tumors, 20brain tumors, 19breast cancer, 20leukemia, 20renal cell carcinoma, 19

354 Index

Microdissection, laser-assisted, 14MicroMED BASIC microwave labstation, 136Microtomy, 67, 68Microwave bulb array, 141, 142Microwave heating, 117

advantages of, 124duration of, 131–133enzyme digestion-assisted, 190, 191high pressure, 133limitations of, 142–144at low temperature, 64, 65, 133mechanism of, 119, 130method of, 124, 126, 127nonthermal effects of, 119, 120role in enzyme studies, 64vacuum-assisted, 69, 133

Microwave heating–EDTA, epitope retrievalwith, 117

Microwave oven, precautions in using, 141,142

Mitotic index, 110Molecular vaccines: see VaccinesMonoclonal antibodies, 1, 2, 37–45

cross-reactivity of, 38, 41, 44, 48, 49, 96production of, 41–44specificity of, 38

Mucin, staining of, 136, 137Multiple antibodies for labeling antigens, 196,

197Multiple antigens, retrieval of, 194, 196

Neuroblastoma, 12Nonbiotin amplification detection system, 99,

100Non-small-cell lung carcinoma, HER-2

localization in, 286, 287Nonspecific staining, causes of, 96, 97, 99Nucleolar organizer-associated region proteins,

209–212quantitative analysis of, 211size, 212, 213

Oncocytic carcinoma of thyroid, 236Osmium tetroxide fixative, 62–64, 161, 165Ovarian carcinoma, 282

HER-2 localization in, 287, 288sialyl-Tn antigen in, 207, 208

p53 (mutant), 10, 12, 75, 86–88, 98, 107, 110,153, 174, 245–259, 284

p53 (mutant) (cont.)antibodies for, 250–253

dilutions of, 253multiple antibodies for labeling of, 256,

257distribution in carcinomas, 256frozen section immunohistochemistry of,

258, 259immunohistochemistry of, 253–256

p53 (wild type), 247, 248, 255, 256microwave heat-assisted retrieval of, 257,

258p73, 249, 250p185 (HER-2), 10, 173, 283Pancreatic adenocarcinoma, 12PAP: see Peroxidase antiperoxidase methodParaffin, properties of, 66–68Paraffin embedding, 65–68

in microwave oven, 67PBS as antibody diluent, 83PCNA: see Proliferating cell nuclear antigenPCR: see Polymerase chain reactionPeriodic acid as antigen retrieval fluid, 76Periodic acid–Schiff’s reaction, 136, 137Peroxidase antiperoxidase method, 89, 98, 99Pheochromocytoma, 282pH of antigen retrieval fluid, 74–79, 82, 83, 96,

104Picric acid, 9Plant tissues, 69Polyclonal antibodies, 34–37

affinity chromatography, 35–37Polymerase chain reaction, microwave

heat-assisted, 224Polyreactive antibodies, 49, 50Pressure cooker, 145

epitope retrieval with, 117, 153method of, 127, 128

Pressure cooker–EDTA method for antigenretrieval, 191

Prion protein, retrieval of, 193–195Progesterone, 9, 78, 129, 145, 153, 279Proliferating cell nuclear antigen, 10, 71, 72,

74–76, 78, 79, 81, 86, 114, 130, 135,139, 255

distribution in carcinomas, 244function of, 11immunohistochemistry of, 242

limitations of, 243–245staining in cryostat sections, 245

Index 355

Prostate specific antigen, 76, 87, 111immunostaining of, 202, 203

Prostatic carcinoma, 111–113, 146, 220HER-2 localization in, 288intraepithelial neoplasia in, 192, 278

PSA: see Prostate specific antigen

Quicgel method, 106

Rapid staining, 136–140of frozen sections, 138, 139method of, 137temperature of, 138

EPOS system, 138, 139RCA technique: see Rolling circle

amplificationRecombinant antibodies, 46Resin embedding, 155–161Resin sections, 5, 155

autoclaving of, 161–163effect of heating on, 161immunostaining of, 158, 159rapid staining of, 163

method of, 166Rolling circle amplification, 92, 93

Scanning electron microscopy, 5microwave heat-assisted, 229, 230

SDS: see Sodium dodecyl sulfateSection thickness, 1Sextant biopsy, 113Sodium citrate buffer, 74–79, 88Sodium dodecyl sulfate, as protein denaturant,

antigen retrieval with, 148–150method of, 149, 150

Squamous cell carcinoma of cervix,HER-2 localization in, 288, 289

Squamous cells of vulva, 236Staining of sections

duration of, 138frozen sections, 138heat-assisted, 136, 137temperature of, 138

Steam-EDTA-Protease methodfor antigen retrieval, 193

Tamoxifen, 270Target unmasking fluid (TUF), 76, 77, 98Telepathology, 25–27

dynamic imagery, 26, 27static imagery, 26, 27

ThinPrep smear, microwave heat-assisted, 278,279

Trastuzumab: see HerceptinTris-EDTA buffer, 98Tris-HCl buffer, 76, 79, 82, 83Triton X-100, 149Trypsin digestion for unmasking antigens, 118,

125, 126, 135, 151, 152, 154TSA-ABC method, 90, 91TUF antigen retrieval fluid, 135Tumor diagnosis, 1Tumors, 3, 6, 10, 12Tyramide amplification technique, 92

Ultrarapid cooling, 65Ultrasonication, 118, 146–148

method of, 148microwave heating with, 189, 190

Urea for antigen retrieval, 76, 98Urinary bladder carcinoma, 88Uveal melanomas, 21, 22, 40

Vaccines, antitumor, 15, 16DNA vaccines, 16HER-2/neu vaccines, 16, 295, 296

genetic immunization, 295, 296protein subunit vaccines, 16virus vaccines, 16

Vacuum embedding, 66Vascular endothelial growth factor, 22–25

in breast cancer, 24immunohistochemistry of, 24

VEGF: see Vascular endothelial growth factorVimentin, 74–76Vulvar lesions, 40, 41

Water bath, epitope retrieval with, 117, 123, 130Wet autoclave method, 145, 146

Zinc sulfate as antigen retrieval fluid, 86