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SUMMARY OF KEY POINTS
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Histology & Its Methods of Study SUMMARY OF KEY POINTS Preparation of Tissues for Study
Chemical fixatives such as formalin are used to preserve tissue structure by cross-linking and denaturing proteins, inactivating enzymes, and preventing cell autolysis or self-digestion.
Dehydration of the fixed tissue in alcohol and clearing in organic solvents prepare it for embedding and sectioning.
Embedding in paraffin wax or epoxy resin allows the tissue to be cut into very thin sections (slices) with a microtome.
Sections are mounted on glass slides for staining, which is required to reveal specific cellular and tissue components with the microscope.
The most commonly used staining method is a combination of the stains H&E, which act as basic and acidic dyes, respectively.
Cell substances with a net negative (anionic) charge, such as DNA and RNA, react strongly with hematoxylin and basic stains; such material is said to be “basophilic.”
Cationic substances, such as collagen and many cytoplasmic proteins react with eosin and other acidic stains and are said to be “acidophilic.”
Light Microscopy Bright-field microscopy, the method most commonly used by both students and pathologists, uses ordinary light and the colors are imparted by tissue staining.
Fluorescence microscopy uses UV light, under which only fluorescent molecules are visible, allowing localization of fluorescent probes which can be much more specific than routine stains.
Phase-contrast microscopy uses the differences in refractive index of various natural cell and tissue components to produce an image without staining, allowing observation of living cells.
Confocal microscopy involves scanning the specimen at successive focal planes with a focused light beam, often from a laser, and produces a 3D reconstruction from the images.
Autoradiography This process localizes cell components synthesized using radioactive precursors by detecting silver grains produced by weakly emitted radiation in a photographic emulsion coating the tissue section or cells.
With either light microscopy or TEM, autoradiography permits unique studies of processes such as tissue growth (using radioactive DNA precursors) or cellular pathways of macromolecular synthesis.
Cell & Tissue Culture Enzyme Histochemistry Histochemical (or cytochemical) techniques use specific enzymatic activities in lightly fixed or unfixed tissue sections to produce visible products in the specific enzyme locations.
Fixation and paraffin embedding denatures most enzymes, so histochemistry usually uses frozen tissue sectioned with a cryostat.
Enzyme classes for which histochemical study is useful include phosphatases, dehydrogenases, and peroxidases, with peroxidase often conjugated to antibodies used in immunohistochemistry.
Visualizing Specific Molecules Some substances specifically bind certain targets in cells.
Immunohistochemistry is based on specific reactions between an antigen and antibodies labeled with visible markers, often fluorescent compounds or peroxidase for light microscopy and gold particles for TEM.
If the cell or tissue antigen of interest is detected by directly binding a labeled primary antibody specific for that ...