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SUMMARY OF KEY POINTS

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
  • Cells can be grown in vitro from newly explanted tissues (primary cultures) or as long-established cell lines and can be examined in the living state by phase-contrast light microscopy.

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 ...

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