Histology (Ch1 Methods)

Microscopy Techniques
1. Light Microscopy
  1. Beam of light is condensed & focused on a specimen
2. Visual Microscopy
3. Electron Micoscopy
  1. TEM (Transmission "")
    1. 60-90nm of tissue
  2. SEM (Scannning "")
    1. Interactions with the beam and the specimen generate
      1. Second electrons which are sent to a monitor
    2. tissue which has been coated with a heavy metal
  3. E- are condensed and focused on a specimen
4. Atomic Force Microscopy
  1. angstrom resolution
  2. silicon-based cantilever focused an infrared laser
5. Described based on their energy source
6. Artifacts
  1. Defects
  2. can occur in any stage
  3. Old or ill-prepared reagents
  4. impure chemicals
  5. Bad timing
  6. knife marks (chatter
Tissue Processing
1. Staining provides contrast to the samples
2. IHC of frozen sections, secondary antibodies
  1. are conjugated to eitheran enzyme or a fluorophore
3. TEM, heavy metals are used
  1. Osmium tetroxide
    1. both fixes and stains membranes
  2. uranyl acetate
    1. stains nuclear material
  3. lead
    1. stains proteins
4. Alternative fixatives to formaldehyde
  1. potassium permanganate
  2. osmium tetroxide
  3. acetone/methanol)
  4. acrolein (tear gas)
Data acquisition techniques
1. Histochemistry and cytochemistry
  1. functional analysis of cells/tissues
  2. Enzyme histochemistry uses enzyme-substrate
    1. reactions to localize enzyme activity
2. Immunohistochemistry
  1. uses primary antibodies directed against an epitope
    1. fluorescently-labeled secondary antibodies
      1. directed against the primary antibody
    2. May detect location of enzymes (not necessarily if they are active)
  2. Direct immunofluorescence employs a fluorescently-labeled primary antibody
    1. Limited Use
    2. useful in flow cytometry
3. Hybridization Techniques
  1. Uses the complementary base-pairing of a radiolabeled
    1. probe to a molecule of mRNA or DNA
  2. Can identify small numbers of nuclei acids
  3. (radio isotopes) 32P, 35S, 3H
4. Autoradiography
  1. radiolabeled monomers
Chemical Basis for Staining
1. Basic dyes interact with anionic moieties
  1. phosphate groups
  2. carboxyl groups
  3. reaction of anionic groups varies with pH
    1. At a pH ~10; all three are sufficiently ionized to react with dye
    2. At pH 5-7; sulfate and phosphate groups are ionized
    3. At pH <4, only sulfate groups react
  4. sulfate groups
2. Stains
  1. Hematoxylin
    1. Not a strict basic dye, similar properties
    2. used with an mordant
      1. Mordant makes it act as a basic dye
    3. used in sequential staining
  2. Eosin
  3. H&E Staining
    1. Loss of information is typical
      1. lipid extraction occurs
      2. Solvents are used for infiltration and curing
      3. The pH of the fixative may hydrolyze some molecules
      4. The aqueous nature may extract glycogen, proteoglycans, GAGs
  4. Acidic Dyes
    1. Eosin, acid fuchsin, aniline blue, orange G
    2. Net (-) charge on the dye
    3. react with cationic groups (acidophilia)
      1. via electrostatic interactions
    4. Basophilic substances
      1. heterochromatin
      2. nucleoli
      3. cytoplasmic components
      4. extracellular materials
      5. identify cytoplasmic filaments, intracellular membranous organelles, and extracellular fibers
  5. Basic dyes
    1. Methyl green, methylene blue, pyronin G, toluidine blue
    2. Net (+) charge on the dye
3. PAS stainscarbohydrate-rich macromolecules
  1. Glycogen
  2. mucus
  3. basement membrane
  4. Periodic acid-Schiff
4. Schiff reagent
  1. Aldehyde groups and basic fuchsin interact, forms red color
    1. Periodic acid-Schiff (PAS) and Feulgen reactions take advantage of this interaction
      1. Feulgen reaction is based on cleavage of purines from deoxyribose
  2. distinct red color

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