Ultrathin cryo-sectioning, immuno - gold labeling

A practical introduction

George Posthuma Viola Oorschot Janice Griffith Elly van Donselaar Despina Xanthakis Suzanne van Dijk Corlinda ten Brink

List of abbreviations:

AB: Antibody APD: Average particle diameter BSA: Bovine serum albumin CV: Coefficient of variation EM: Electron microscope FA: Formaldehyde FCS: Fetal Calf Serum GA: Glutaraldehyde IgG: Immuno-globulin G LE: Labeling efficiency LN2 Liquid nitrogen MC: Methyl cellulose Mwt: Molecular weight PAG: Protein A-gold PB: Phosphate buffer PBS: Phosphate buffered saline PIPES Piperazine-N,N-bis(2-ethanesulfonic acid HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid PFA: Paraformaldehyde PVA: Polyvinyl alcohol PVP: Polyvinylpyrrolidon RT: Room temperature TA: Tannic acid UA: Uranyl acetate

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Table of Contents

1. General introduction ......................................................................... 3 2. The specimen .................................................................................... 5 3. Chemical fixation .............................................................................. 5 3.1. Formaldehyde .................................................................................... 6 3.2. Glutaraldehyde ................................................................................... 6 3.3. Acrolein ............................................................................................ 7 3.4. Mixtures of aldehydes ......................................................................... 7 3.5. Other conditions affecting fixation quality ............................................... 7 3.5.1. pH / buffers ....................................................................................... 7 3.5.2. Temperature ...................................................................................... 7 3.5.3. Osmolarity ......................................................................................... 7 3.5.4. Duration of fixation ............................................................................. 8 3.5.5. Fixation procedures ............................................................................ 8 3.6. Storage and shipping of fixed specimens ................................................ 8 4. Support and cryoprotection .............................................................. 9 4.1. Extracellular support ........................................................................... 9 4.1.1. Tissue ............................................................................................... 9 4.1.2. Cells ................................................................................................. 9 5. Mounting and freezing .................................................................... 11 5.1. Specimen carriers ............................................................................. 11 5.2. Mounting ......................................................................................... 11 5.3. Freezing and storage of frozen specimens ............................................ 11 6. Sectioning ....................................................................................... 12 6.1. The environment .............................................................................. 12 6.2. The ultra cryo-microtome .................................................................. 12 6.3. The specimen block .......................................................................... 13 6.3.1. Size and shape of a specimen block: Trimming ..................................... 13 6.3.2. The consistency of the block. ............................................................. 14 6.4. The knife ......................................................................................... 15 6.4.1. The art of making glass knives. .......................................................... 16 6.5. Knife alignment and approaching the block face .................................... 17 6.6. Sectioning ....................................................................................... 19 6.6.1. Compression .................................................................................... 20 6.6.2. The operator .................................................................................... 20 7. Section retrieval (Pick-up) .............................................................. 20 7.1. Mounting of LM-sections .................................................................... 22 7.2. Storage of thawed cryosections .......................................................... 23 8. Immuno-labeling. ........................................................................... 23 8.1. How many gold particles can be expected? ........................................... 24 8.2. Some background information ............................................................ 24 8.3. Immuno-labeling strategies ............................................................... 25 8.4. Immuno-double labeling .................................................................... 27 8.5. The labeling procedure ...................................................................... 27 8.6. Background reduction ....................................................................... 28 8.7. The immunoreaction ......................................................................... 29 8.8. Controls .......................................................................................... 29 8.9. Rinsing ........................................................................................... 30 8.10. The marker ...................................................................................... 30 8.11. Stabilization of the immune reaction ................................................... 30 8.12. Double labeling procedure ................................................................. 31

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8.13. Section Light Electron Microscopy (SLEM) labeling. ................................ 32 8.14. Immuno-fluorescence labeling ............................................................ 32 9. Contrast and support ...................................................................... 33 10. Carbon-Formvar coated grids ......................................................... 34 11. Protocols ........................................................................................ 35 11.1. Diamond knife cleaning ..................................................................... 36 11.2. Fixation ........................................................................................... 39 11.3. Carbon-Formvar coated grids ............................................................. 42 11.4. Correlative light electron microscopy (CLEM) ........................................ 45 11.5. Gelatin embedding ............................................................................ 47 11.6. Immuno-labeling .............................................................................. 50 11.7. Ionizer (Static Line II) usage .............................................................. 53 11.8. Mounting & Freezing ......................................................................... 54 11.9. Loops and hairs preparation ............................................................... 56 11.10. Protein A-Gold (Tannic acid procedure) ................................................ 58 11.11. Silan coated microscope slides ........................................................... 63 11.12. Section Light Electron Microscopy (SLEM) Immuno-labeling ................... 64 12. Recipes ........................................................................................... 66 12.1. BSA 10% (100 ml) ........................................................................... 66 12.2. Formaldehyde 16% (500 ml) .............................................................. 66 12.3. Formvar solution for grids (100 ml) ..................................................... 67 12.4. gelatin 2% in 3cm Petri dish (100 ml) .............................................. 68 12.5. Gelatin 12% for cell support (100 ml) .................................................. 68 12.6. Methyl cellulose 2% (200 ml) ............................................................. 69 12.7. Methyl celluloseuranyl acetate pH 4 (10 ml) ...................................... 69 12.8. PBS 10X pH 7.3 (2.5 lit.) ................................................................... 70 12.9. PBS/glycine 0.02 M ........................................................................... 70 12.10. PHEM, 0.2M buffer pH 6.9 (100ml) ...................................................... 71 12.11. Phosphate buffer 0.2M pH 7.4 (100 ml) ............................................... 71 12.12. Pipes 0.2M buffer pH 7.3 (200ml) ....................................................... 72 12.13. PVP - Sucrose pH 10 (20 ml) .............................................................. 72 12.14. Sucrose 2.3M (100 ml) ...................................................................... 73 12.15. Uranyloxalicacetate PH7 (100 ml) ....................................................... 73 12.16. Uranylacetate 4% pH4 (10 ml) .......................................................... 74 12.17. Suppliers ......................................................................................... 75 13. Addendum ...................................................................................... 76 13.1. Protein A- G- L affinities .................................................................... 76 13.2. Rousselot gelatin .............................................................................. 77 13.3. Short incubation schedule. ................................................................. 78 14. References Tokuyasu technique. .................................................... 79

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General introduction 1.In this course we are primarily concerned with methods for localizing proteins within cells using high-resolution electron microscopy. This approach is often referred to as EM immunolocalization, EM immunolabeling, or immuno EM. We are also concerned about achieving the best possible preservation of cell ultrastructure, and of the antigens that we will label with antibodies. But before we get into the details of the course contents, lets take a brief look at how we got to where we are today, and why we prefer the methods well present in this course. Most of what we know about the interior ultrastructure of cells comes from transmission electron microscopy of epoxy resin sections. Unfortunately, these sections do not work well for EM immunolabeling because the surface of the epoxy section is generally not permeable enough to gold markers (Stierhof et al., 1989). Nor is it possible to immunolabel vitreous cryosections because they have to be kept cold and dry for imaging. While it is possible to do immunolabeling of freeze fracture replicas (Rash et al., 2004) this method requires specialized equipment that is not commonly found in most EM labs. Today the two most common methods for EM immunolabeling are the Tokuyasu technique, and on-section labeling of methacrylate resin sections. Both methods owe some debt of gratitude to Fernandez-Moran, who showed many years ago (1952) that processing samples at low temperatures by cryo-immobilization, cryo-substitution, or progressive lowering of temperature deydration (PLT technique) improved the preservation of cell ultrastructure. On-section labeling of methacrylate resin sections evolved mainly in two directions: the Lowicryls and LR White resins. (Kellenberger et al. 1980; Weibull et al., 1980) picked up on the PLT idea of Fernandez-Moran and combined that with methacrylate resin formulations (Lowicryl K4M and HM20) that could be polymerized at low temperature. ( Newman et al. 1982: Newman and Jasani, 1983)) introduced the LR White resin and showed that it would accept both immunoperoxidase/diaminobenzadine (DAB) and colloidal gold markers for immunolabeling. Initially, the LR White processing did not involve cryoprocessing, though nowadays it is sometimes used in conjunction with cryo-fixed and cryo-substituted samples, or LR Gold resin is used. While we believe that these on-section labeling methods, especially with the Lowicryls, are useful in some situations, we prefer the speed and convenience of the Tokuyasu method, as well as the fact that it tends to be less damaging to the cells when done properly.

Tokuyasu realized early on (Tokuyasu, 1976) that infiltration of fixed samples with sucrose would prevent the ice crystal damage that was such a problem for the cryofixation methods of that era. This method also had the very significant advantage of not requiring dehydration prior to sectioning. It is well known, and has been convincingly demonstrated (see chapters in Crang and Komparens, 1988) that dehydration is one of the main culprits for producing the fixation artifacts that have plagued EM studies for years.

Figure 1.1 :Graphic representation of the standard processing steps in the Tokuyasu technique.

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Thus, the Tokuyasu technique is mindful of a basic rule of immunolabeling: that the less you change the sample, the better the chance of recognition by an antibody. Sucrose does not enter living cells under normal circumstances, but it does not fix cells either. Thus, the sucrose infiltration step is necessarily preceded by a fixation step. In the classical Tokuyasu method the sequence of events is as follows: fixation support sucrose infiltration freezing sectioning retrieval (storage) immuno-labeling final contrasting and support (Figure 1.1) Here at the Utrecht University Medical Center we have been using and improving the Tokuyasu technique for EM immunolabelling since its earliest days. Jan Willem Slot worked with Tokuyasu in the early seventies in the lab of Jonathan Singer at the University of California, San Diego, and the first cryo-ultramicrotome was installed here at Utrecht in 1977. In these early days Slot and his colleagues continued to modify the basic technique, often in concert with Tokuyasu himself and with Gareth Griffiths who established a similar facility at the European Molecular Biology Laboratory (EMBL) in Heidelberg, Germany. Several other technical developments were emerging in parallel that would have a big impact on improving this technique and making it easier to perform. Manufacturers were making better cryo-microtomes for one thing, and there were improvements in diamond knives at about the same time. It is worth mentioning that the idea of using diamond knives for sectioning was also an idea of Fernandez-Moran (Fernandez- Moran, 1953). Another important step forward for EM immunolabeling was the development of colloidal gold particles. Inspired by the results of Jrgen Roth and colleagues (Roth, Bendayan et al. 1978) on resin embedded sections, we started to use gold particles and developed methods to produce uniform gold particles of different sizes (Slot and Geuze 1985), which is of paramount importance for localization of different antigens in the same section. The high electron density of the gold markers allowed more contrast in the sections. With Gareth Griffiths (Griffiths, McDowall et al. 1984) modification of Tokuyasus adsorption staining method, an elegant staining procedure was developed that results in a beautiful delineation of membranes and contrast differences in cytoplasm and nuclei which we still routinely use. In 1995, Willisa Liou in our lab started to evaluate the damaging effect of the sucrose solution that had been used routinely for retrieving and thawing of the cryo-section. The outcome of that study, in which the entire EM group participated, was a relatively simple modification in the method of cryosection retrieval, but it had a staggering effect on the preservation of the ultrastructure in the sections (Liou, Geuze et al. 1996). These are a few highlights from the many improvements that were introduced during the last 30 years and that have made the Tokuyasu technique the reliable and powerful localization tool which it is now (Slot and Geuze 2007). This manual is an attempt to give an impression of how the technique is routinely performed in our laboratory. We update it on a regular basis and this would not have been possible without the valuable help of the people in the lab. The 2011 CMC course manual is the result of about 210 years of cumulative experience with immuno-electron microscopy. It is used during practical courses, but it does not pretend to describe all possible alternatives found in other laboratories, nor do we try to give a complete overview of the literature. In the first part we describe the method and give some basic background without being too elaborate. More in depth information can be found in some excellent books given in the literature section. The second part will exist of step-by-step protocols with a tips and tricks section. In the course we also discuss and demonstrate high pressure freezing and freeze substitution and their possibilities. In 2010 Kent McDonald from the University of California, Berkeley joined our course as a lecturer and added the part of high

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pressure freezing and freeze substitution to this manual and changed the text into real English for which we are very grateful. Because many people were expressing interest in the cryo-sectioning and immunolabeling skills that had been acquired in our group, we started annual courses in 1999 with the collaboration and support of Leica Microsystems, Vienna. The course is 10 days long, which is long enough to receive practical, hands-on training, and we keep the number of students small to ensure that everyone has adequate opportunity to learn the material. In the past more than 200 people have completed the course and we hope that they enjoyed it as much as we enjoyed working with them. We plan to continue this series in the future as a means to communicate further developments in this field to our colleagues.

Utrecht, 18-6-2012

George Posthuma Viola Oorschot Janice Griffith Elly van Donselaar Suzanne van Dijk Despina Xanthakis Corlinda ten Brink

The specimen 2.Only the best specimen is good enough for your experiments.In fact the specimen is the most important item of the entire procedure. When animals are concerned: gender, stress, drugs, diet, parasites and transgenic changes may affect the quality of the tissue you are looking at in an unexpected way. In the case of cultured cells (transient) transfections, the culture conditions, infections (yeast, bacteria, mycoplasm), drugs, viruses etc. will also affect the quality of what will be seen in the electron microscope. Do not waste time on suboptimal specimens.

Chemical fixation 3.The aim of fixation is to maintain the structural integrity of tissues and cells so that they can be studied in the electron microscope. Immuno-electron microscopy adds some additional demands:

1. The substances under investigation (antigens) must remain recognizable for an antibody

2. The antigens should remain at their natural site of occurrence. 3. The antigens should stay (or become) accessible for the immuno-reagents.

Often these demands are in conflict with each other. Preservation of the (ultra)structure and natural distribution of the antigens need a decent and quick fixation but that same fixation may mutilate the antigenic site in such a way that the antibody cannot recognize it. Furthermore it may intensify the matrix compactness due to cross-linking of cellular components and will hamper penetration of immuno-reagents. Fixation is lethal to cells due to the fact that most proteins stop functioning. Inevitably the cell becomes leaky for ions and non cross linked (usually low Mwt) molecules and ions. When cells and intracellular compartments are still surrounded by a more or less intact membrane some extraction will occur. However when a

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frozen section is picked up all the non cross linked molecules are in direct contact with a buffer solution and will be extracted quickly. In general, antigens attached to cellular components like membranes or the cytoskeleton will remain in the cryosections. When working with these naturally bound antigens, mild fixation may be an advantage since many cytoplasmic components are extracted from the sections and the accessibility for immuno-reagents will be increased. When quantitative retention of antigens (especially soluble) is required a strong fixation is essential. Some organisms will need special treatment to achieve a good fixation. For instance yeast has an extremely tough cell wall that needs to be rendered permeable for fixatives. Other organisms like C. elegans will live happily ever after in fixative and different measurements like cryo-immobilization and freeze substitution have to be taken. The most frequently used fixation agents for immuno-cytochemistry are aldehydes. These compounds are toxic. They are very reactive and volatile, in particular acrolein. Therefore it is important to handle them only in a fume-hood with gloves. A very useful survey the chemistry of fixation can be found in Griffiths book (Griffiths, Simons et al. 1983)

Formaldehyde 3.1.Formaldehyde (FA, methanal, O=CH2, Mwt 30) is a gas that easily dissolves in water. Usually it is sold as a powder which in fact is a polymer (n>8) called paraformaldehyde. In water it forms also polymers, which can be turned into monomers with OH- ions and heating to 63C. For Electron microscopy it is of great importance to work with monomers. Small molecules can easily enter cells and be there in time to minimize the changes in the dying cell. Formaldehyde has only one aldehyde group that can react with free amino groups Cross linking occurs in time due to the formation of methylene bridges. The formation of methylene bridges between molecules is enhanced by longer fixation times, higher pH (8 - 8.2) and by using higher FA concentrations (> 5%) (Fox, Johnson et al. 1985). When high concentrations are used directly on cells or tissue, osmotic events might occur. For instance the osmolarity of 8% FA in 0.1M phosphate buffer is 1300, which is more than 4 times the physiological osmolarity. However the effect of osmolarity is limited because the formaldehyde molecules will react with the cells constituents and hence contribute less to the osmolarity of the fixation solution. When osmotic events are observed the initial fixation can be done at low concentrations (2%) of FA, followed by stepwise increase in concentration. Its low Mwt allows the FA molecule to penetrate quickly into specimens. It provides a good morphology especially after prolonged fixation. (8 72 hour). This fixation process is partly reversible and prolonged storage of tissue in buffers without fixative may result in a loss of content and structural integrity. In the Tokuyasu technique it used to be difficult to preserve the ultrastructure in FA-fixed thawed cryosections, but the introduction of new pick up procedures has improved that substantially. In general FA is a better fixative than GA for successful immuno-reactions. Extraction of materials and less cross-linking favour penetration of immuno-reagents and the antigens themselves are less denaturized.

Glutaraldehyde 3.2.Glutaraldehyde (GA, pentanedial, O=CH-(CH2)3-CH=O, Mwt 100) has two reactive aldehyde moieties, which mainly react with lysine residues of proteins and will crosslink most proteins very efficient, either intra or inter molecular. For electron

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microscopy the application of monomeric GA is a prerequisite because long polymers will not enter cells quickly. Hence always use EM grade GA. The fixation is irreversible and its penetration is relatively slow. The morphology is very well preserved. GA is a strong fixative in immuno-cytochemistry. The ultrastructure is stably preserved, but antigen denaturation often occurs. During fixation a good buffering system should be used, otherwise the pH might be lowered 2-3 pH units.

Acrolein 3.3.Acrolein (propenal, O=CH-CH=CH2, Mwt 56) has one reactive aldehyde group that mainly reacts with lysine. Furthermore the double bond between the carbon atoms may play a role in cross linking. Its fixation is irreversible and its penetration into tissues and cells is relatively fast. Acrolein is mostly used in combination with GA or FA.

Mixtures of aldehydes 3.4.

A mixture of GA and FA and or acrolein can be used in various combinations. The effectiveness of such mixtures is not an addition of reactivity of both components. Both aldehydes will compete for amino groups. Therefore the mixture may result in stronger fixation than from 2% FA, but cross-linking may be less than when only 0.2% GA is used

Other conditions affecting fixation quality 3.5.

pH / buffers 3.5.1.The pH should be maintained within a physiological range during the fixation process. It is known that the aldehyde fixatives lower the pH when they react. Therefore the buffering capacity of the fixative solution should be strong. Omit PBS and use a stronger buffer system like 0.1M phosphate buffer. Some commonly used buffers like TRIS may be less suitable during fixation. Our concern is that the amino groups will quench the aldehyde action. Furthermore substances can be added to the buffers that favor the preservation of specific cellular components like cytoskeleton elements (PHEM buffer). Extra precautions should be taken when the specimen has been fixed with uranium salts like uranyl acetate, which is often used in freeze substitution. These salts react with constituents of phosphate buffer and give rise to unwanted precipitations. A HEPES or Pipes buffer should be used in these cases.. Suitable buffers are phosphate buffer (0.1M, pH 7.4), HEPES (0.1M, pH 7.4) or PIPES (0.1M, pH 7.4).

Temperature 3.5.2.The initial fixation should be performed at room temperature or even at 37C. At those temperatures the structural integrity of the cytoskeleton is better preserved. Furthermore the diffusion of the fixative into the tissue or cells is faster and the actual cross-linking will be more effective. After the initial fixation the specimens can be stored for longer periods of time at 4 C.

Osmolarity 3.5.3.A physiological osmolarity for a fixative would be 360 mOsm. Some fixatives like FA can increase the osmolarity to a great extent. For example 8% FA in a 0,1M phosphate buffer has an osmolarity of 1300 mOsm. However the effect on cells is

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not as profound as these figures may suggest. First, the aldehydes are presumably uncharged when added to the cells and therefore they can pass freely through biological membranes. Next they start to react rather quick with amino moieties and therefore do not contribute as much as anticipated based on the molarities to the final osmolarity. However when rather slow reacting substances are used like FA, osmolarity might be taken into account (see also 3.1).

Duration of fixation 3.5.4.The time during which specimens should be fixed depends on the size of the specimen, the nature of the antigen and the used fixative, its concentration and the temperature. Usually, 2 hr at room temperature is sufficient for fixation with GA containing fixatives. FA (without GA or acrolein) fixation is usually performed overnight. Prolonged fixation does not necessarily improve the ultrastructural preservation, but may affect the antigenicity of antigens under investigation. Furthermore prolonged fixation may harden the specimen and will affect the sectioning properties.

Fixation procedures 3.5.5.It is important to disturb the material to be fixed as little as possible prior to fixation. For instance do not wash or trypsinize cells when it is not necessary. In particularly ice cold media or even PBS will influence the cells morphology. It is usually better to leave them under growing conditions and add fixative. Also the fixative should be at temperatures of 20 -37 C, at least at the beginning. Tissues from animals should be fixed by whole body perfusion when possible. After an initial flush with PBS (RT) to remove the blood, the fixative (RT) is introduced into the (anesthetized of) animal via an artery or the left ventricle of the heart. Biopsies should be immersion fixed as soon as they are excised from the body and cut into thin (

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Support and cryoprotection 4.

Extracellular support 4.1.Embedding of specimens in a matrix similar to the specimen serves several purposes:

1) Cell suspensions can be concentrated at will and the blocks are easy to prepare and handle.

2) It allows orientation of specimens (correlative LM-EM) 3) It improves the conditions for sectioning substantially: it fills extracellular

spaces thereby equalizing the consistency of cells and extra cellular spaces. 4) In sections of non embedded cell suspensions and to lesser extent in loose

tissues without embedding cell profiles float around individually when thawed on the pick up solution. When embedded in a meshwork they behave more coherently, which is particularly important for serial sectioning or correlative light and immuno-electron microscopy.

In general we use gelatin as supporting matrix. Gelatin comes in many flavours. The source (pig-, cow-, fish- etc. bones or skin) and the way it was treated and purified determine the stiffness of the final gel. Usually this is expressed as Bloom figure: the higher the Bloom figure the stiffer the gel will be (when the same percentage is used). The relatively high mol weight of the denaturised collagen prevents it from entering fixed cells. We use food quality gelatin, which is compatible with all the buffer systems we have tested till now. A good alternative for gelatin is low melting point agarose, especially when part of the specimens has to be osmium fixed and embedded in a resin. After sectioning gelatin can be removed at 37 from the section while agarose or fixed albumin cannot.

Tissue 4.1.1.In figure 4.1 a schematic drawing is made of a simple way to prepare appropriate blocks of gelatin supported tissue. For tissue a microscope slide is completely covered with a layer of Parafilm, whereas on either end an additional 6 layers of Parafilm are placed. The specimen (in 37 gelatin) is placed on the Parafilm and another with Parafilm covered slide is placed on top (Figure 4.1-1). Next this is placed on ice and the gelatin is allowed to solidify (Figure 4.1-2). The top slide with Parafilm is removed and the gelatin slab with tissue

can be cut in appropriate blocks (Figure 4.1-3,4). Next the block are transferred to 2.3 m sucrose, mounted on a specimen holder (Figure 4.2-5, 6,7) and frozen.

Cells 4.1.2.Cells are rinsed twice with 37 gelatin and spun down in an Eppendorf tube (Figure 4.2). The tube is placed in ice and the gelatin is allowed to solidify. The bottom of

Figure 4.1: embedding of tissue in gelatin

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the tube containing a high concentration of cells in gelatin is cut off, removed from the bottom of the Eppendorf tube and cut into 0.5x0.5x 0.5 mm blocks with a razor blade (Figure 4.2-1,2,3,4). Next the blocks are infused with sucrose and mounted on a specimen holder (Figure 4.1-5,6,7) and frozen. Cryoprotection It is important to prevent ice crystal formation in the specimens during freezing. First of all ice crystals would damage the ultrastructure, but besides that, good cryosections can only be cut from vitreous frozen blocks. This makes freezing of fresh biological material a real problem. Ice crystal formation can only be prevented when freezing occurs within milliseconds. Heat conductance becomes the limiting factor with the result

that in practice vitreous freezing is restricted to the very surface layer of, at best, ~10 m. This can be extended to a few hundred microns under high pressure conditions. Working with chemically fixed tissue, as we do in the Tokuyasu technique, ice crystal formation is simply circumvented by infusing sucrose into the tissue. Sucrose is a rather inert, hydrophilic compound that easily diffuses through the cellular membranes after fixation. It does not seem to affect the fixed tissue, even not at the highest possible concentration of 2.6M. Tokuyasu introduced the sucrose infusion and later it was shown by Griffiths et al. (Griffiths, McDowall et al. 1984); and McDowall et al.(McDowall, Chang et al. 1983) that sucrose solutions of 1.8M or higher vitrify when they freeze, no matter how slow the freezing takes place. The entire specimen needs to be infiltrated with 2.3 M sucrose otherwise sectioning becomes very difficult and ice crystal damage may occur. We use an overnight infiltration in a rotating carousel at 4 C. In sucrose infused tissue, the cellular constituents and the extra cellular gelatin probably also contribute to the vitrification hence lower sucrose concentrations may be sufficient for proper cryoprotection. This can explain why Tokuyasu had good results with regard to cryoprotection when he used sucrose concentrations below 1.8M. He recommended initially 0.6 - 1.5M for different tissues, depending upon their protein density. Sucrose infusion serves other purposes as well. Around 1980 we found that infusion of the tissue blocks with 2.3M sucrose improved the sectioning characteristics substantially (Geuze and Slot 1980) for any tissue and cell preparation that we worked with. This high concentration of sucrose renders a plasticity to the frozen blocks that, allows cutting of very thin sections that are still flat and glossy at -120. Another possibility to improve sectioning is the usage of a mixture of polyvinylpyrrolidone (PVP) and sucrose especially for loose tissues , cells with large voids (Tokuyasu 1989) and cells grown on filters.

Figure 4.2: Embedding of cells in gelatin, sucrose infiltration and mounting.

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Mounting and freezing 5.

Specimen carriers 5.1.The aluminum specimen carriers (pins) should fit perfectly in the microtome otherwise the sectioning will be very irregular. Usually they are provided with the microtome by the manufacturer; however ordinary rivets seem to perform just as well and are much cheaper. The sucrose infused block should be glued with sucrose to the pin. The top of the pin is roughened, with a sharp metal point. The same can be achieved with sandpaper, but the grains will damage the diamond knife when they are not removed properly. The small groves improve the attachment of the specimen to the surface. The surface needs to be clean, hence we sonicate them in acetone to remove any greasy material and metal remnants (do not touch the surface of the pins with your fingers!!) Check with binoculars whether the pins are perfectly clean and free of metal remnants. Again such remnants may ruin a precious diamond knife. The pins can be reused after removal of the specimens and sucrose by sonicating them in distilled water and acetone respectively.

Mounting 5.2.The specimen blocks are removed one by one from the 2.3M sucrose by means of forceps or a tiny wire loop and placed immediately on the table of a clean pin. Be careful not to touch the table surface with anything except the specimen, in particular not with your fingers. Remove most of the excess sucrose with a piece of filter paper, but leave enough to glue the block at the basal side to the specimen holder (Figure 4.2-7) Mount the specimen in a good position for sectioning. Sometimes the specimen has to be sectioned in a specific orientation, for instance cross sections of muscle fibers or kidney medullar tubules. It may be advisable in that case to shape the blocks in such a way that orientation still can be recognized, for instance a long edge parallel to the muscle fibers. The final trimming of the blocks can then be done right before the mounting, so that you still remember which side of the block has to be the cutting face. The mounting procedure should be done as quickly as possible. Water evaporates from the specimen during air exposure, and then sucrose concentration becomes so high that sucrose crystals will be formed. For that reason we perform these steps under a binocular in the cold room. If there is no cold room available, keep the blocks cool and put the pins in a plastic block (not metal, because you will get water from the air on your pin) in ice and and freeze your blocks one by one.

Freezing and storage of frozen specimens 5.3.The mounted blocks, glued by the remaining sucrose to the specimen holder, are then put in LN2 or in the cryochamber of the ultra cryo-microtome. The sucrose needs to remain clear after freezing. If it turns milky the sucrose concentration is too low and water crystals have formed. This will affect the sectioning properties of your specimen If the crystals have not damaged your specimen, renewed infiltration with real 2.3 M for 4 hr on a rotator will solve this problem. When the specimens are relatively big, copper or, to a lesser extent, aluminum specimen holders cool down much quicker than the specimens and crevasses may emerge between the holder and the specimen. This will not occur when the specimen and pin are frozen slowly by freezing them in the (at least 80 C) cryo-chamber of the microtome. Crevasses will also emerge when the table of the pin has not been

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cleaned properly (grease). As a result during sectioning chattering, irregular sectioning will occur, in the worst case the specimen may break away from the pin. When this happens it is possible to remount the specimen, since freezing is no longer a crucial step. Pick up the specimen and put it back in 2.3 M sucrose (4 C), leave it there for 30-60 min and remount. We use Sanbio cryotubes for storage, its lid fitted with two holes. The holes in the lid prevent internal pressure build up and form also a grip for firm tweezers when opening or closing the cryotubes. Each cryotubes contains an aluminium block, which prevents it from floating. Seven 8 mm holes, drilled in the metal block form seven numbered compartments, each of which can contain several pins with specimen blocks, we often use colour coding (using a standard felt pen) for the pins as well. In this way many specimen blocks can be stored in one LN2 container. Frozen specimens (on specimen holders) can be stored in LN2 for months or even years. Prolonged storage in LN2 may freeze-dry the specimen surface to a certain extent. If this renders sectioning more difficult, renewed sucrose infiltration and remounting the specimen solves this potential problem.

Sectioning 6.The quality of ultrathin (cryo) sections is depending many factors:

1. The environment 2. The ultra cryo-microtome 3. The specimen bloc 4. The knife. 5. The Tools 6. The operator

The environment 6.1.The environment in which the microtome is placed is of great importance to the way it will function. It should be in a vibration free room without large air flows with a controlled humidity. Most of the vibration problems are solved when the microtome is placed on a standard table that comes with the microtome. If this is not sufficient the microtome can be placed on an active anti vibration device. Air that flows around the cryochamber will disturb the stability of the atmosphere inside the chamber and this will result in temperature changes and excessive ice formation in and around the cryobox. Humidity in the room is a factor that affects the buildup of ice and the static conditions in the cryochamber. For the Tokuyasu technique a humidity of approximately 50% is good. When frozen sections from vitrified specimens (without 2.3 M sucrose) are cut for cryo-electron microscopy the humidity needs to be as low as possible and a special box around the microtome is required.

The ultra cryo-microtome 6.2.The current ultra-microtomes have reached a technical degree of perfection, that allow regular feed with an accuracy of 1 nm, which means that 15-200 nm sections have become reality. Furthermore the sectioning speed can be controlled very precise from 0.1 mm/sec to 100 mm/sec. The combination of this extremely accurate and precise movement with precise temperature regulation in the cryochamber of specimen, knife and cooling gas makes cryo-sectioning a much

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easier job as compared to 10 years ago. Despite this degree of perfection maintenance is very important. Spindles and motors ware off and greases do age. We have our microtomes serviced once a year thoroughly and give them a quick checkup 6 months later. Special attention should be given to the LN2 pump. When the pumping is not regular, e.g. the pump is always pumping with the same speed during operation, the sectioning will be irregular. Before you start sectioning always check whether the flow and pressure are normal. We judge that by turning the pump on without the tube toe microtome attached to the pump. If this is OK, but still the amount of LN2 flowing into the chamber is not sufficient, the tubing is probably clogged with ice and needs to be cleaned. We keep the pump during the week in the LN2 and dry it during the weekend. When drying is not sufficient the pumping mechanism requires cleaning. Last but not least the dewar needs attention: in 3-6 months there will be a lot of ice crystals floating in the LN2, these will eventually stop the pump from functioning properly.

The specimen block 6.3.The way a knife interacts with a specimen depends on the size, shape and consistency of a block.

Size and shape of a specimen block: Trimming 6.3.1.When you start to cut ultrathin sections the block needs to be flat on the front side, small enough and have a rectangular (square) form with straight edges.

First the front is flattened using a good glass knife or a diamond trimming tool. At high temp (-100C) with a high speed (100 mm/sec) relatively thick sections of 200 300 nm can be cut. The largest protrusions should be on the lower side of the block, otherwise during the sectioning a large piece of your block may chip away (Figure 6.1-A, D). The total surface should be like a tiny mirror. White dots in the section indicate the presence of plain sucrose and will affect the sectioning. During the trimming put the ionizer close to your knife edge at 9, it will blow away most of the dust you generate. At this point a

semi-thin section should be Figure 6.1: trimming of a specimen block. A: with a glass knife, B: with a diamond trimming tool.

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picked up, stained and evaluated in the light microscope (correct region in the tissue, enough cells ? etc).

After the area of interest has been selected, the size of the block is determined. It is possible to cut ultrathin sections of large blocks (2x2 mm) but keep in mind that with an increasing size of the specimen block the forces necessary to actually make the section become larger and the sectioning becomes more troublesome. As a general rule: the smaller the block the easier it is to section. When sections become thinner the necessity for smaller dimensions becomes more evident. (Al-Amoudi, Dubochet

et al. 2003; Matzelle, Gnaegi et al. 2003). With the latest microtome models the block size can be measured. Usually sizes of 200-500 m work quit well.

The shape of a block is also important for the final result. For resin sections usually a trapezoid form is used. This allows an obvious distinction between subsequent sections and makes them easy to

separate. Frozen sections are cut dry and have more interactions with the knife surface. For cryosectioning a square (Figure 6.2-B) or a rectangular (Figure 6.2-C) block is much more practical. In block no C the long side is 1.5 times longer than the short side which makes it easy to judge the degree of compression of the section. With 30% compression the final result is a square section. Avoid an orientation as shown in Figure 6.2-D.

The sides need to be straight and the resulting block needs to be rectangular. This can be realized by using a diamond trimming tool, the corner of a glass knife or a razor blade (and a steady hand) . Trimming with a diamond trimming tool or a glass knife is quite similar.

A diamond trimming tool (we prefer a 45 cutting angle with 20 sides) cuts both at the front and at the sides, so it will give straight edges. With a trimming tool one side is trimmed 50-100 m deep, the knife is retracted, the tool moved to the other side of the block and again 50-100 m deep is trimmed away. Be aware of the angle of a trimming tool: when 50 m deep has been removed with a tool with 20 angle on the sides it also cuts away 18 m of the front of the block. Next the block is turned 90 and the same routine is performed. Turn the specimen back to its original position and remove any debris from the surface by cutting one or two 50 nm sections. With a glass knife only one side can be used and it is necessary to turn the specimen 4 times 90.

The consistency of the block. 6.3.2. Extremely soft blocks are very difficult to cut. During the sectioning process these will have extreme compression which results in a much thicker section than the feed

Figure 6.2: different specimen block forms. A: common for resin sectioning, B and C: suitable for cryosectioning, D: less suitable for cryosectioning.

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indicates (see Figure 6.3 ). Hard (brittle) materials are often also difficult to cut because chipping will easily occur as indicated in the lower panel of Figure 6.3. In cryosectioning the hardness of the block can be controlled with the temperature: the lower the temperature the harder the block. Last but not least the uniformity of the consistency is also affecting the sectioning characteristics of a block. The more differences in consistency the more difficult it is to section, especially when soft and hard portions are parallel to the knife edge. Hence embedding in gelatin for cryosectioning will

improve the sectioning. In contrast to resins, gelatin will not enter the interior of a cell, so when there are large voids in the specimen, that can not be reached by gelatin, it might be advantageous to add in addition to the sucrose a high molecular weight substance like polyvinylpyrrolidon to improve the consistency after freezing.

The knife 6.4.A knife has a number of given characteristics. First of all there is the material the knife is made of. For electron microscopy only two materials have been used

routinely to produce ultrathin sections: glass and diamond. A glass knife is made out of a special kind of glass with a knifemaker, usually with an angle of approximately 45 Diamond knives are prepared from natural gem quality (or even better) diamonds and come in many sizes and shapes. The top angle can be as sharp as 15, however those knives are very vulnerable. The sharper the knife angle the less compression a knife will give (Matzelle, Gnaegi et al. 2003; Al-Amoudi, Studer et al. 2005) Further more the radius (Figure 6.4 R)of the very edge of the

Figure 6.3:Sectioning properties of soft (upper panel) and brittle (lower panel) specimens. Soft specimens will yield more compression, while brittle specimens will have many crevasses or even chip away during sectioning.

Figure 6.4: Knife edge properties: The facet of a knife is grinded with a certain angle in this case 35and has a top radius (R) . Both determine the sharpness of a knife. The way the surface is treated after grinding determines the he properties of its surface.

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knife will also play an important role during sectioning. A blunt knife (large radius) will never cut. Moreover the surface of the knife that is in contact with the section is extremely important. The surface of a glass knife is obviously glass, whereas the surface of a diamond knife is one of the best kept secrets of the diamond knife manufacturers. After grinding a diamond to the desired dimensions, the polishing material is removed and the surface is treated in such a way that it gets its final properties. The message is that for each specific application there is an appropriate surface available. Especially when the sectioning is performed dry, the surface of the diamond determines whether sectioning will be a success or not. For cryosectioning of 2.3 M sucrose infused specimens an almost ideal surface has been created by Diatome . To use the maximum benefits of such a surface good cleaning is imperative

The art of making 6.4.1.glass knives. The preparation of a glass knife starts by selecting good glass strips from which they are made. The glass should be tough and preferably without consistency differences in the glass (Pittsburgh glass). When a new batch of glass strips arrives, the dimensions and quality should be carefully checked. The width of the strips must be constant without distortions on the lateral sides of the strips. If the strips do not meet your requirements send them back to the manufacturer.

The perfect glass knife has a straight or slightly concave cutting edge. Its top angle is

approximately 45 and does not show any serrations in a binocular. In the 45 plane the breaking lines are barely visible.

The best way to make knives is according to Tokuyasus balanced break method (Tokuyasu and Okamura 1959) using a Leica knife maker (KMR3). In theory a

Figure 6.5-A: Braking of glass knives according to the balanced brake method). B: The final scratch is positioned as close as possible to the diagonal of the square and is broken, this will yield an approximately 45 knife. C: When the scratch is not on the diagonal the top angle of the knife will be close to 80

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perfect knife is made by breaking a glass rod in two equal parts, next each part is broken in two equal parts etc., till squares of 2.5 x 2.5 cm are produced (Figure 6.5-A). Because equal forces have been applied to the glass this balanced breaking method yields squares, with freshly straight edged surfaces. The sharpest knives will be obtained when the breaking occurs almost exactly on the diagonal of the square (Figure 6.5-B). Glass will break towards the nearest surface an when the scratch is away from the diagonal (Figure 6.5-C) the top angle will be close to 80 (!), which will make sectioning a kind of scraping. Furthermore the crack should develop slowly using moderate forces and should be perpendicular to the upper surface of the square. Fast breaking will result in an elevated stress line that will affect the knife

edge in a negative way (Figure 6.5-A). To meet all these requirements the midpoints of the upper pins of the knife maker must necessarily be positioned at equal distances from the scoring line. The support pins at the lower surface of the glass should be positioned exactly beneath the scoring line (Figure 6.6-A). This can be checked by breaking several twin blocks. If the breaking plane shows the same aberration repeatedly, the position of the scratch should be adjusted since the position of the support pins is set by the manufacturer. To be able to fracture the squares along the diagonal the position of the square is carefully

examined after each fracture and adjusted when necessary. If the counter piece is hardly visible and the sharp edge is made out of the freshly broken plane, the knife is probably quite good (Figure 6.6-B, always check with a binocular). With older knifemakers positioning of the square needs some extra attention and adjustments. Knives with a convex edge (Figure 6.6-B3) or with elevated stress lines (Figure 6.6-A fast) should not be used.

Preparing knives as described above may seem laborious, but the invested time is not idly spent, since the ideal knife will save much time and annoyance during sectioning. In our lab we store the knives in a plastic box for several months.

Knife alignment and approaching the block face 6.5.Once the block has been trimmed, the trimming knife is replaced by a sectioning knife. This should be at sectioning temperature, absolutely clean and without any frost. If the block is still in the microtome upper and lower part of the block will have equal distances to the knife edge (alignment north-south). When a previously

Figure 6.6: Knife dimensions after braking. A: the stress line (arrow) will be very prominent after fast braking as shown in the drawings of the upper part of the knife. B1:Knife with a straight edge, B2: knife with a concave edge, C3:knife with a convex edge. The latter is not suitable for sectioning.

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trimmed or sectioned block is used this may not be the case as shown in figure 6-7 A and B were the backlight illumination is used to visualize the gap between knife and specimen. When the reflection on the specimens surface is identical on lower and upper side of the specimen, there is no need for correction otherwise the front

Figure 6.7: Knife alignment North-South. When the specimen surface is not equidistant to the knife edge, while moving up and down, the reflection on the specimen surface changes from wide to small. In a cryo-microtome this cannot be corrected, so a new blockface needs to be prepared

Figure 6.8 : Knife alignment East-West. When the blockface is not parallel to the knife edge in east west direction, the knife must be (very carefully) turned

Figure 6.9 Aproach: When the backlight is turned on and the knife is in the vicinity of the trimmed face a bright white area can be observed on the block face. This is the reflected light from the back of the knife and the specimen surfce(A). When the knife approaches this reflection becomes smaller (B) and finally it changes to a color (C)

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needs to be equalized by cutting a few sections on a trimming knife since a cryo-ultra microtome does not have a segment arch. Using previously trimmed block the lower edge of the block needs to be aligned with the knife edge. After trimming the edge of the sectioning knife usually is not parallel to the specimen. When the reflection of the backlight on the block is not perfect rectangular as in Figure 6.8- A&C, the knife should be rotated in such a way that the reflection is a perfect rectangle (Figure 6.8-B). When block face and knife edge are perfectly aligned the knife needs to be positioned so close to the block that the next stroke of the microtome will result in a section. Again the back light is very useful. While moving closer and closer to the block the reflection becomes smaller and smaller (Figure 6.9 A >B) till it is no longer white and becomes colored (Figure 6.9-C). Once blue only one or two 50 nm steps forward are necessary to cut the first section.

Sectioning 6.6.The ideal section has a perfectly flat surface, has a uniform thickness, no knife marks, is not folded and has no compression. As with many things such perfection only exists in fairy tales. When the knife encounters the specimen no one knows exactly what happens. However the knife has a finite sharpness which means that it starts to push into the specimen thus cutting a section. Depending on the hardness of the specimen (Figure 6.3 and Figure 6.11) some block deformation will occur. The section will be pushed onto the surface of the knife and will be compressed due to the friction and in a dry environment charge will build up. In reality the initial section thickness as defined by the feed (T1 in Figure 6.11) will be up to 3 times more due to these processes (T2 in Figure 6.11). In resin sectioning this also occurs, however the friction and static problems can be minimized by using a trough with water that acts as a lubricant and also prevents static charges to build up. In cryo-sectioning

Figure 6.11 Interactions between knife and specimen. During sectioning the knife cleaves the specimen. The lower surface of the section will encounter friction on the knife surface and compression will occur further more static charges (--) will be generated

Figure 6.10: Charges on a knife surface can be regulated with an ionizer.

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many attempt have been made in the past to find something equal to water. Thus far without success. A fluid has not been found yet, that remains liquid at low temperature and has the same surface tension as water, a prerequisite for obtaining a floating section. In cryosectioning a different lubricant is used( Figure 6.10 : --- on knife and section). During sectioning static charges build up on the knifes surface. The amount of charge depends on the weather (humidity) the person (clothing) the kind of specimen and most important the knifes surface. Till the introduction of the controllable ionizer this static charges could not be regulated and you were lucky if everything worked. With an ionizer the amount of charge can be carefully controlled (in most cases) and the charge on the knife and section make the section hoover above the knife surface thus reducing compression to a reasonable level (30%) and allowing the section to be manipulated using an eye lash. With the correct settings and a bit of luck, it is possible to make long ribbons till even 2 cm long!

The sectioning speed is also an important factor. In the past the text on the box of a diamond knife stated: tested at 1mm/sec and that speed was what most people used. The combination of the blocks consistency, the knife, the charges and the speed will ultimately determine whether the sectioning will be satisfactory or not. It is worthwhile to vary the speed when the sectioning does not work. We have varied in the past between 0.2 mm/s till 80 mm/s! It will not damage your knife and might be the solution to your sectioning problem.

Compression 6.6.1.Compression is expressed as: 100- (section length/block surface length)*100 . (i.e. a percentage). During the sectioning the section pressed onto the knife surface, thus compressing the section. When the angle between knife surface and block face is larger, this effect will increase. The second factor is Figure 6.11). The section sticks to the knifes surface, whereas at the same time it is pushed away from the knife edge along the knife surface by the rest of the section. The first problem can be minimized by using knives with a small top angle (Al Ahmoudi et al , 2003), the second by toning the knife surface with an ionizer and varying cutting speeds. Unfortunately in cryosectioning compression cannot be avoided.

The operator 6.6.2.The operator is a very important factor: you need to get acquainted with your machine in combination with your specimens, knives, environmental circumstances etc. Besides skills, the person hem- (or her-) self influences the sectioning because the operator acts as a source or drain of static electricity. Well insulating shoes in combination with synthetic fabrics will render sectioning virtually impossible. We advice cotton fabrics and sometimes a grounding bracelet (like people that handle electronic components).

Section retrieval (Pick-up) 7.Once the cryo ultra-microtome produces relatively flat, minimally compressed sections, they have to be retrieved from the knife. Under the right conditions modern

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cryo ultra-microtomes, like the Leica Ultracut S or T/FCS and more recently the UC7/FC7, regularly produce ribbons of sections. Usually the sections are guided with an eyelash on top of a wooden stick away from the knife edge. The ribbons are detached from the knife edge and moved aside somewhere down on the diamond knife or on the epoxy between knife holder and diamond knife (NB: do not transfer to the metal part of the knife, the sections cannot be retrieved properly) The size of the ribbons should not exceed half the diameter of the wire loop used to retrieve the sections. Sections have a tendency to jump towards the pickup solution when the solution temperature is not correct. During their journey to the pickup solution sections wrinkle. Therefore it is advisable to aim for the center part of the drop, to prevent this as much as possible. When 2.3 M sucrose is used sections need some space to spread out over the pick-up drop during thawing. Usually not more than 4 or 5 sections should be included.

When enough sections are collected, stop the sectioning and move them away from the knife edge and pick them up. The ionizer should be turned off during section retrieval. A wooden stick with a stainless steel loop (loop diameter of 2.5 mm, wire diameter 0.3 mm) is dipped into a pick-up solution. Retracting the wire loop quickly out of the pick-up solution, in a direction perpendicular to the plane of the loop, will result in a large drop. Slow withdrawal, sideways, will decrease the drop size. The size of the drop is important since a larger drop will take longer to freeze. We have currently two different pick-up solutions in our laboratory.

The first solution we started with is 2.3 M sucrose in 0.1M PB. The wire loop filled with a sucrose droplet is introduced into the cryochamber. While looking through the binocular, the droplet is brought to a position where you can see it in the binoculars, yet as far as possible away from the sections. Usually some smoke (freezing water vapor) is emerging from the droplet. Once it stops smoking press the droplet gently on top of the gathered sections with the center of the droplet. Do not try to pick up too many sections otherwise the surface of the droplet becomes overcrowded. Looking through the droplet the sections are visible, and sometimes it is possible to see the sections (over)stretch, soon after that the sucrose solution is frozen. Remove the droplet from the cryochamber and allow the sucrose drop with sections to thaw. Some people help the thawing by gently breathing on the drop. The effect of that is questionable and when uranyl or fixatives are added to the retrieval solution (see below) it may be a risky habit. After thawing, press the droplet with the section side down on a Formvar carbon-coated grid. To check the quality of the sections, the sucrose is removed by floating one grid on distilled water. After approximately 5 minutes the grid is removed from the water, air dried and viewed in the electron microscope. If the sections are satisfactory, the other grids can be stored as described in section Storage of thawed cryosections.

The second solution we use is a 1:1 mixture of 2.3 M sucrose in PB and 2% methyl cellulose in distilled water which is prepared just before use. Prepare this mixture fresh from cold solutions (4 C) and mix the constituents very gently (or use a rotator) and keep it on ice during sectioning. Mixing at room temperature will give white deposits, which is probably due to withdrawal of water from the methyl cellulose solution by the sucrose before the mixing is completed. We started using this second pick-up solution in 1996 after we found a vastly improved ultrastructure in the sections picked up this way (Liou, Geuze et al. 1996). Since then this is the

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standard way to pick up sections in our lab. The viscosity of this solution is much lower and it freezes much quicker. The freezing is obviously crystalline. It is easy to follow under the binoculars how it starts freezing at the edge (white rim) in the corner of the loop. This is the moment to pick up he section. Wait longer and the drop is completely frozen and the sections cannot be retrieved. With the MC/UA solution the sections do not stretch on the droplet during pickup or thawing, so what you see is what you get!! This implies that only the shiny flat sections give optimal results. It needs some experience to use this pickup method successfully, but it pays off at the end. The fine structure is much better as compared to sections retrieved with 2.3 M sucrose.

In most cases 2.3 M sucrose or sucrose/methylcellulose are suitable pick-up solutions, but 2-4% polyvinyl alcohol can be used also. When delicate lipid rich structures are involved these pick-up solutions are not sufficient to retain lipids in a cryosection. An additional on-section fixation is necessary to preserve lipid-rich structures and the lipids in membranes. For these purposes Willisa Liou used a mixture with final concentrations of 2% methyl cellulose and 2% uranyl acetate in distilled water (Liou, Geuze et al. 1997)). The sections must be of outstanding quality , thin (50 nm) and flat. She also had some success by thawing cryosection of fresh, cryo-immobilized material in pick-up solutions containing mixtures of uranyl and aldehyde fixatives. In other words she did the complete chemical fixation on the sections (Liou, Geuze et al. 1996).

Mounting of LM-sections 7.1.When the semi-thin 200nm thick sections are used for immuno-fluorescence (IF) studies it is also an advantage when the sides have been trimmed, however these section are rather ridged in comparison to ultrathin sections and it is not absolutely necessary. 2.3M sucrose is a good pickup solution, the sections stretch slightly, which enhances the accessibility of antigenic determinants. The z resolution of a semi-thin section is 2-3 times better than an optical confocal section (in theory 0.2m but I reality 0.5-0.7 m). For IF the sections are placed on 3-aminopropyltriethoxysilan coated microscope slides. With a diamond pen we scratch lines of ~1.5 cm perpendicular to the length axis of the slide. One slide can carry 3 scratches at ~2cm distance from each other. After the scratching, make sure that all glass splinters are removed with a paint brush or compressed air. 3 or 4 pick-up drops (either 2.3 M sucrose or 1.15 m sucrose/1% MC) with semi-thin sections can be placed alongside a scratch (scratch and sections at the same side of the slide). Two different specimens needing identical immuno-staining can be placed on each side of the scratch. For different antibodies, sections have to be placed along different scratches. A fine wax pen is used to demarcate the area with sections around a scratch. The wax line prevents the incubation drops from pulling away from the scratch area. The wax should be allowed to dry at least a day. The sections with sucrose can be stored in a cool place for several days prior to immuno-labeling.

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Storage of thawed cryosections 7.2. After the sections have been put on top of the grid, they can be stored in two ways. When the grids are used within the next 24 hrs they can be transferred to 2% solid gelatin plates (sections facing the gelatin). To prevent drying out the 3 cm plates are stored in Petri dishes at 4 C in a closed container. Keep the gelatin cool during sectioning. Second we introduced the storage of sections while still covered by the pick-up drop of sucrose/methyl cellulose (Griffith and Posthuma 2002). The grids remain attached to the stickered microscope slide on which they are prepared. In that position the grids with sections are stored in a closed microscope slide storage box at 4 C. It was found that drying under these conditions did not affect the ultrastructure or immuno-reaction. For many molecules this is a perfect storage method; however one should always test your antibody on a stored and freshly cut section. Small or difficult to fix molecules may diffuse out of the section during this storage.

Immuno-labeling. 8.

As soon as biological research in the 1940s became aware of the fact that an antibody (AB) can be used to mark the molecules to which they were directed (hence called antigens) they became an indispensable tool in science. In theory it is a simple procedure: First an antigen is purified, next it is injected into an animal of a different species and nature does its job. The animal will recognize the antigen as a foreign molecule and will produce antibodies which will recognize their antigen out of millions of other molecules. Another widely used approach is genetic labeling. In this case the protein of interest is tagged genetically with a relative small protein like hemaglutinin, green fluorescent protein or with the smallest tag available at the

Figure 8.1: Immunolabeling with ProteinA-Gold

Figure 8.2: Immunolabeling with IgG Gold

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moment: tetracystein(Gaietta, Giepmans et al. 2006). The latter would be in theory the most suitable tag, but there are as yet no antibodies available and Reash reagents in combination with DAB cytochemistry must be used for detection in the electron microscope. When ABs are available, they can be used to visualize antigens on gels, blots, tissues, cells and (ultra-thin) sections with unprecedented accuracy provided they are coupled to a suitable marker. To be able to see the antibodies in the electron microscope an electron dense marker is necessary. At first antibodies were coupled to enzymes that could produce an electron dense precipitate (after osmium tetroxide fixation). Although extremely valuable, this technique has its limitations: the reaction products are diffusible, sometimes difficult to discern from the surrounding tissue elements and difficult to quantify. Visualization of more than one antigen in a specimen is not possible. For immuno-electron microscopy a very small discrete electron dense marker would be ideal. Faulk and Taylor (Faulk and Taylor 1971) introduced the gold particle as a marker for the electron microscope. Gold particles can be made in different sizes (Slot and Geuze 1981) and can be coupled to a wide variety of biological active molecules : enzymes, antibodies, toxins, lectins, protein A and many more. Since that time immunogold labeling is the most widely used technique to visualize molecules in the (transmission or scanning) electron microscope. For obvious practical reasons the primary antibody is not coupled to an electron dense marker (availability of antibodies, shelf life). The primary antibody is visualized by means of Protein A coupled to gold (Figure 8.1) or an IgG (Figure 8.2).

How many gold particles can be expected? 8.1.Before an immuno-labeling study is performed, one should always realize that there should be enough molecules present to be visualized. In a thin section only a very small part of the cell is visible. A 50 nm thick section of a standard 10x10x10 m cell only represents 0.5% of the total volume or 0.3% of the plasma membrane! Furthermore molecules will change during their lifetime in a specimen: they are trimmed, glycosylated, phosphorylated, polymerization takes place, etc. Moreover, the antigenic determinants can be destroyed by fixation and may not be accessible to antibodies in the specimen thus further reducing the number of available molecules. The highest reported labeling efficiency (LE) is approximately 10%(Griffiths and Hoppeler 1986), but often it is less than 1 %. When this standard cell has 30,000 molecules on its plasma membrane, in the electron microscope between 0.9 (LE=0.01) and 9 (LE=0.1) gold particles can be observed along the membrane.

Some background information 8.2.Molecules bind to each other, either very weak (no binding or even repulsion) or very tight, the quality of this binding is described by a dissociation constant or Kd. A Kd < 10-8 describes a strong bond between AB and antigen whereas a Kd > 10-3 means that the two are not tightly bound to each other. In the Bjerrum Plot in Figure 8.3 different theoretical plots are displayed. The curve with the solid circles represents a genuine AB-antigen binding where, at a concentration of 5x10-8 mg/ml, 95% of all

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the available antigen molecules are bound to an AB. The other curves represent the binding of that same AB to other molecules. At a concentration of 10-4 mg/ml every available antigen molecule is bound to an AB, but at the same time the AB binds to

other molecules as well due to electrostatic or hydrophobic interactions: background. The solution to the background problem is to dilute the AB to the appropriate concentration and block possible nonspecific binding sites with molecules that do not bind to the specific AB but do bind to molecules in the specimen. A blocking agent should be selected depending on the nature of the

nonspecific binding.

When antigens are very abundant it is often quit

obvious whether the correct AB concentration is used. However when that is not the case, statistics will give a clear answer whether the labeling is specific or not as recently described in a brief yet comprehensive tutorial by Mayhew. (Mayhew 2005)

Immuno-labeling strategies 8.3.In general, 3 different strategies can be discerned: whole mount immuno-EM, Pre-embedding labeling and post-embedding labeling.

Whole mount immuno-EM is in fact the fastest method. Specimens are adhered to grids or cultured on grids, (mildly fixed) and labeled. When the labeling is performed without any detergents present in the incubation media it is most likely that only the outside of a specimen will be labeled. In this procedure the entire cell is present on the grid and later in the electron microscope. Therefore only rather thin, non-electron dense, specimens can be used in 60-120 KV electron microscopes. In Figure 8.4 an Enterococcus has been immuno-labeled for a surface antigen. The same technique can be used to visualize antigens on viruses, blood platelets and the rims of flat cells. Immuno-labeling of the

Figure 8.3: Bjerrum plot of antibody-antigen binding. Small Kd :strong binding, larger Kd weaker binding

Figure 8.4: Whole mount immunolabeling.EM Picture of an Enterococcus adhered to a Formvar-Carbon coated grid. After fixation a surface antigen has been labeled with primary antibody and protein A-Gold. Final staining with MC/UA. Bar=200 nm

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interior of a specimen is possible when the specimens are treated with detergents like saponin or Triton X100. This will generate small pores in the surrounding

membranes and (part of) the cytoplasm and making the antigens accessible.(van Dam and Stoorvogel 2002)

Closely related to the previous technique is the pre-embedding technique. The specimens are immuno labeled before they are embedded in a resin and sectioned. This is a very useful technique when the distribution of surface antigens is studied. To access the interior of a specimen (cell) the plasma membrane has to be opened prior to the labeling procedure. This can be achieved by detergents or repeated freeze-thawing, which are rather mild treatments. Cells can also be opened with an osmotic shock or by mechanical forces like scraping them from their support. Even after such a harsh treatment, many organelles are still intact but the cytoplasm is no longer present. The absence of cytoplasm during

labeling increases the availability of antigenic determinants of cytoplasmic tails from membrane associated molecules. It has been used for instance to visualize molecules on synaptic vesicles (De Camilli, Harris et al. 1983). In Figure 8.5 one of the coat proteins (AP1) present at the Trans Golgi Network of HepG2 cells is labeled with 5 nm gold particles. In this particular

case the cells were fixed wit 1.0% formaldehyde for 5 min and scraped from the Petri dish and embedded in 1% agarose. Small blocks were incubated overnight consecutively with antibodies, gold probes and afterwards embedded in EPON. The last approach is post-embedding immuno-labeling. After the specimens have been supported by a resin, methacrylate or ice, the sections are cut and the labeling is performed on the

section. With a thickness of 70 nm most organelles will be cut open and their interior is exposed to antibodies. It is obvious that the way in which

Figure 8.5: Pre-embedding labeling. Cells were permiabilized and immunolabeled for AP1and protein A-gold. Thereafter the cells were embedded in EPON sectioned and stained with UA and lead. Arrow: clathrin coat, bar=200 nm

Figure 8.6 : Immuno-double labeling of a Hela cell, which was transfected with lung surfactant protein C (SPC). Labeling: anti SPC > protein A-gold 10 nm and anti PDI (resident ER protein, mouse antibody) >rabbit anti mouse> protein A-gold 5 nm. Bar =200 nm.

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the specimens were treated will influence the labeling efficiency. Not only the fixation will influence the number of available antigenic determinants, but also the chemicals in the embedding procedure will destroy or mask epitopes. The immune reaction takes place at the very surface of the section and the availability of epitopes is quite different for each embedding method. Sections from specimens embedded in an epoxy resin have a rather smooth surface which means that there are not many epitopes available. The surface of hydrophobic methacrylates (Lowicryl HM20, LR gold) resembles that of the epoxy resins, the more hydrophilic methacrylates (Lowicryl K4M, LR white) have a rougher surface. Thawed frozen sections usually will give the best LE because the antigenic determinants have not been exposed to organic solvents and embedding media. Moreover their surface is rough and antibodies can penetrate into the section (depending on the matrix density of the organelle). Unfortunately gold particles ranging from 5 to 20 nm hardly penetrate into a section due to their size and charge. (Stierhof and Schwarz 1989) This problem can be tackled(partially) by using ultrasmall gold particles ranging from 0.8 to 2 nm. Due to their small size, these particles are hardly visible in the electron microscope and need to be increased in size by means of silver or gold enhancement (Yi, Leunissen et al. 2001).

Immuno-double labeling 8.4.Due to the particulate nature and the well-defined sizes of gold markers it is possible to label more than 1 epitope on a specimen.(Figure 8.6 and Figure 8.7) In theory the number of different epitopes that can be labelled is only limited by available gold particles. However, with an increasing number of immune-oreagents the greater the chance of background labeling will be. In practice double labeling is frequently used and occasionally also a triple labeling.

The labeling procedure 8.5.During the immuno-labeling procedure the sections are incubated over a series of different drops by floating the grids on top of the drops, sections facing the drop. For most rinsing steps drops of 50-100 l are used which can carry up to 3 grids at once. For antibody and immuno-gold solutions we use 5-10 l for each grid. The drops are placed on a clean and flat surface. This is easily achieved by using a Parafilm sheet that is adhered to a glass plate by some distilled water. The cover sheet of the Parafilm is removed step by step while the incubation proceeds, so that the surface is kept clean. The grids can be transferred by wire loops or by forceps. Using the latter, less of the incubation fluid is transferred to the next drop especially when non capillary tweezers are used, which renders the washing steps more efficient. The adhering fluid may decrease the concentration of the immuno-reagent which are usually in 5 l drops. To transfer a minimum of excess fluid (approximately 0.5 l), pull the grids sideways and gently from the drops. Excess adhering fluid can be removed with filter paper, but be careful: never allow the section side of the grid to dry. Drying will damage the ultrastructure and a dry section surface tends to be sticky to all kinds of proteins including immune-reagents. This will result in high background labeling. The backside of the grid should stay dry throughout the procedure. If a grid accidentally sinks and one wants to save it, wash it in distilled water. Then dry the back side by carefully (but quick) wiping with filter paper and let it float on clean distilled water before entering the incubation procedure again.

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For a typical immuno-labeling of the sections, transfer the grids over the incubation media mentioned successively. Routinely all media are buffered by PBS until the washing steps in distilled water. Other buffer systems can be used as well. One should be aware of the fact that uranyl will give precipitates with buffers that contain much sodium. Copper grids should be replaced by nickel or even golden grids when very long incubation procedures are followed or aggressive chemicals like in in situ hybridization buffers are used. The incubations are at ambient temperature, unless indicated differently. A detailed protocol is given in the protocol section.

Background reduction 8.6.Both the support film and the sections will bind immuno-reagents due to hydrophobic, charge mediated etc interactions. This problem can usually be solved by covering these binding places with proteins. In the immuno-labeling for Tokuyasu sections the sections are placed on 2% solid gelatin in a 3 cm petri dish. The petri dish (with lid) is placed in 40C stove and kept there for 20 min. The melted gelatin will cover many protein binding places and at the same time dissolve the gelatin in between cells and pickup solution. After rinsing away the excess of gelatin, a smaller, differently charged molecule like BSA is used to reduce the background even more. There is a wide variety of blocking solutions: cold water fish skin gelatin, ovalbumin, 1-5% (fetal) calf serum, dissolved fat free skimmed milk powder, diluted goat serum, or 0.1-1% acetylated BSA (Aurion). The physical properties of the antibody and section determine whether background reduction is successful and it worthwhile to try different blocking agents or a combination thereof.

A different source of background might be free aldehyde groups, which I theory can also bind immuno-reagents. Those are incapacitated with an amino acid like glycine, but also Tris will do the job. Sometimes we use 0.1% -1% NaBH4. Sodium borohydrate is very unstable (Tokuyasu, 1997) and it should be used immediately after being prepared, or even refreshed once during an incubation period which is usually 5 min. The hydrogen radicals will quench free aldehyde groups, reduce double bonds in molecules induced by (GA) fixation and thus may restore antigenicity. Additionally the tiny hydrogen bubbles may render the sections more open which results in an improved penetration of the immuno-reagents during the incubations. In case of immuno-fluorescence NaBH4 treatment takes away auto-fluorescence induced by GA.

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The immunoreaction 8.7.Immunoreagents are usually diluted in 1% BSA/PBS, but other blocking proteins or buffers can be used as well. The dilution has to be worked out for each particular antibody. The grids are floated on 5 l drops. They dry out easily, in particular when long incubation times are used, so the drops should be kept in a humidified atmosphere (wet tissue under a square Petri dish). In general, the duration of the immuno-incubation is not very critical when high affinity antibodies are used. The minimum time we use is 20 min at room temperature. The necessary time might be longer when the concentration of antibodies is low, the temperature is low or the Kd is high. Furthermore one should realize that antigens might be extracted from the thawed sections during incubation. That may happen to molecules that do not react with aldehydes, like membrane lipids, but also soluble proteins may escape easily when the fixation is weak (Posthuma, Slot et al. 1987). In such cases it is better to keep the incubation short. On the other hand, proteins that are integrated in membranes or bound to the cyto-skeleton will not easily escape and labeling of these may be favored by longer incubation periods, during which soluble compounds may be extracted, so that penetration into the section may improve. When the incubation is extended to the next day, we usually keep the grids overnight at 4 C in a moisture atmosphere. In that case the antibody should be further diluted (~10 times) to prevent that non-specific binding becomes a problem.

When the final marker does not react with the primary IgG it is necessary to use a bridging step. For example goat IgGs or many mouse monoclonal antibodies will not react with protein A. Be aware of the fact that background may increase with each step and that the distance between epitope and gold particle increases after each step. There is one major advantage of using one (or more) bridging steps: more epitopes are visualized because the AB can penetrate into a some parts of a section thus increasing the labeling efficiency. (Slot, Posthuma et al. 1989).

Controls 8.8.To check for non-specific labeling by the primary antibody, the bridging antibody, or the PAG or IgG probes, sections should be incubated in parallel and treated with an identical incubation procedure as the experimental grids

Checking for non-specific labeling induced by the primary antibody can best be performed by processing and labeling tissue or cells that are similar to the specimen, but are lacking the antigen. This is a feasible control in all studies on transfected gene products or when knock outs are available. The second best control is to perform a labeling with the preimmune serum. This should not give any gold particles when a similar dilution is used. It is important to supplement these tests with electrophoresis and immuno-blotting. Unfortunately the results do not always match perfectly: antigen molecules in solution may have different binding characteristics than molecules in sections. Another test would be to mix AB and antigen prior to the incubation. We often found huge clusters on our sections after incubation pre-absorbed AB solutions.

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Rinsing 8.9.After each immuno-reagent the grids have to be rinsed. The grid is removed from the droplet sideways with tweezers and usually only 0.5 l is left. Quickly transfer the grid to the next (rinsing) drop of approximately 75 l. 3 rinses will in theory dilute the antibody 150 x 150 x 150 = 3,375,000 times which is usually more then sufficient to abolish any remaining activity.

The marker 8.10.The best visible markers for electron microscopy are gold particles bound to IgGs , protein A, lectins etc. They are manufactured in a wide range of sizes (0.8 40 nm) and allow double, triple or even quadruple labeling. Of course all of these markers can bind nonspecifically and should always be diluted to the proper concentration in a background reducing solution. We prefer Protein A-gold over any other molecule since the protein A can be easily purified, gives very reproducible results and is more precise since only one PAG particle can bind to 1 Fc part of a primary or bridging antibody. Furthermore it allows double labeling when the primary antibodies are from the same species, because the Fc biding site for protein A is destroyed by a brief glutaraldehyde fixation. However PAG has also some disadvantages. It does not bind to the all the IgG subclasses of every species. Due to its relative small size (Mwt 42 kD) as compared to IgG (Mwt 176 kD) it may not recognize IgGs just beneath the section surface since the charge of the gold usually prevents the gold particle to enter the section.

Stabilization of the immune reaction 8.11.When primary antibody, bridging antibody and marker are in place, it is useful to stabilize this reaction with a fixation step. The laws of physics tell you that although firmly bound, each of the immuno reagents can (and will be) detached in time. To prevent that a 1% glutaraldehyde fixation is used. This is particularly useful in an immuno-double labeling. Besides stabilizing the immune reaction it also destroys the protein A binding site on the Fc portion of the IgG, so it will not be recognized in a double labeling procedure by the second PAG particle. The GA treatment can be a problem when the second immuno-reaction is GA sensitive. Fortunately we noticed that this on-section GA fixation is often not as bad as one would expect for antigenic sites that are killed when GA is used for the initial cell. There are two possible explanations for that. First, GA sensitivity is often a matter of penetration barriers that are created by the cross-linking effect of GA. It may well be that these barriers cannot be formed anymore in a weakly fixed section that has gone through a series of incubations. During these incubations many molecules have been extracted, that could be part of these barriers. Second, if GA cannot be used for initial fixation, one usually fixes with FA. Since both aldehydes react primarily with the same groups, largely amines of protein molecules, it may well be that the GA reaction is rather mild with proteins in sections that are prefixed with FA.

When GA fixat