Yuanjing Liang

Department of Histology and Embryology, Capital Medical University

Cell: 13520893699 Office: 83911448Email: y3liang@126.com

Introduction

1. Four primary tissue types in our body2. HE staining.3. Electron microscopes, electron

dense/electron lucent.

Histology is the study of tissue of the body and of how this tissue are arranged to constitute organs.

Hierarchy of Anatomical Organization

Molecules (simple and macro) Sub-cellular (organelles) Cells (cytology) Tissues (histology) Organs (organology) Organ systems Organism Supra-organismal:  Societal / Ecological

Types of tissue • Epithelial tissue• Connective tissue• Muscular tissue• Nervous tissue

• Understanding tissue structure at levels not visible to the unaided eyes, including three dimensional relationship among the biochemical constituents.

• Understanding the relationship between tissue structure and function.

• Establishing a basis for learning histopathology, which involves the relationship between abnormal tissue structure and functional defects.

• Providing basis for treating diseases and injured tissues.

FixationDehydration, clearing, infiltration, embedding SectioningStaining

1. FixationThis is to avoid tissue digestion by enzymes.Chemical fixation: formalin, osmium tetroxide Fresh frozen

2. Dehydration, clearing, infiltration, embedding

These steps replace the water within the cell with a more rigid material to permit thin sectioning. Dehydration: ethanolClearing: xyleneEmbedding: paraffin, plastic, resin

3. SectioningCryotome MicrotomeUltra-microtomes

Hematoxylin-eosin staining (HE)Tissues stained by acid dyes (e.g., eosin) are termed acidophilic and are usually basic in nature, thus acid dyes preferentially bind to cytoplasm and extracellular fibers.Tissues stained by basic dyes (hematoxylin) are termed basophilic and are usually acid in nature, thus basic dyes preferentially bind to nuclei, heterochromatin, rough endoplasmic reticulum.

4. Staining

1. Light microscopy (LM)

With the light microscope, stained preparations are usually examined by means of light that passes through the specimen.

Resolution human eye-200 μmlight micoscope-0.2 μmelectron microscope-0.002 μm

Compound bright-field microscope Dark-field microscope: objects that deflect light into the objective lens appear bright on a dark background. Useful to examine crystals.Phase contrast microscope: transform invisible differences in phase retardation into visible differences in light intensity. Often used with unstained specimens or cells in vitro. Polarizing microscope: polarizing filters placed on both sides of specimen. Birefringent items (e.g., collagen, striated muscle) appear bright on dark field.Fluorescent microscope: the fluorescent substances appear brilliant on a dark background.Interference microscope: allows quantification of tissue mass.

Confocal scanning microscope: uses laser for fluorescence (permitting small region of excitation) and computers to generate image. Allows for optical sectioning of the specimen and 3-dimensional reconstruction.

2. Electron microscopy (EM)

Transmission electron microscope (TEM)

TEM permits visualization of the internal ultrastructure.

Fixation: glutaraldehyde for proteins and osmium tetroxide for phospholipids.

Dehydration followed by infiltration and embedding in resin.

Sectioning: 10-100 nm thick sections made with diamond or glass knife on ultramicrotome. Sections are floated onto copper mesh grids and electron beam passes holes in grid

Dark areas of an electron micrograph are usually called electron dense, whereas light areas are called electron lucent.

Scanning electron microscope (SEM)

SEM permits visualization of the surface ultrastructure.

3. Basic principles of histochemistryHistochemistry is used to demonstrate specific enzymes or minerals in contrast to more generally dye (histological) staining.

4. Some important biologic substances & classic methods for detecting them

IonsLipids: Sudan black, oil red ONucleic acids: Feulgen reactionCarbohydrates: (glycogen) periodic acid-Schiff reaction

5. Enzyme histochemistryAcid phosphataseDehydrogenasePeroxidase

6. ImmunohistochemistryImmunohistochemistry (IHC or immunocytochemistry (ICC)) is the use of antibodies (Ab) to localize specific gene products in specimens.

Direct

Indirect

Identification of rat bone marrow stromal cells by BrdU labeling and detection

Marrow stromal cells were stained with a mouse anti-BrdU antibody, detected with an FITC-conjugated anti-mouse secondary. (A) Most nuclei of cells were stained green by FITC, ×200. (B) B is the same location of A under a light microscopy, ×200.

7. In situ hybridization

In situ hybridization is method of analyzing the tissue distribution of particular nucleotide sequences in DNA and RNA.

8. Cell, tissue & organ cultures

This methods are used to study living cells and tissues without the interference of the organic homeostatic mechanisms.

9. Cell fractionationThis procedure is used to isolate and collect cellular components in quantity to study their contributions to cell function.Homogenization, centrifugation, purification.

10. Electrophoresis

One-dimensional gel electrophoresis: An electric field applied to a solution of protein, or other large molecules, causes them to move in a direction and at rates reflecting their net charge.

10. Electrophoresis

Isoelectric focusing

10. ElectrophoresisTwo-dimensional gel electrophoresis: is used for separation of complex protein mixtures by the independent parameters of isoelectric point and molecular weight.

11. Blotting & Electrotransfer

Target molecules migrate from the gel to the membrane. Then the membrane is incubated with solution of labeled antibody or complementary nucleic acid sequence to reveal the position and relative amount of the molecules of interest.

12. Genetic technologyIsolating genes and DNA fragmentsDNA sequencing: encompasses biochemical methods for determining the order of the nucleotide bases in a DNA oligonucleotide.

12. Genetic technology Copying genes and DNA fragments DNA cloning: refers to the procedure of isolating a defined DNA

sequence and obtaining multiple copies of it in vivo. Polymerase chain reaction: the DNA thus generated is itself used

as a template for replication. This sets in motion a chain reaction in which the DNA template is exponentialy amplified

1. The slides are flat pictures of 3D objects 2. Correlate structure to function3. Make chart

Parenchymal organHollow organ

Tissue: 4 primary tissue typesCell: amount, distribution shape nucleus cytoplasm (organelles) function

1. Four primary tissue types in our body2. HE staining.3. Electron microscopes, electron

dense/electron lucent.