Mindmap
Histology (Ch1 Methods)
Inhalt der Mindmap
Histology (Ch1 Methods)
Microscopy Techniques
- Light Microscopy
- Beam of light is condensed & focused on a specimen
- Visual Microscopy
- Electron Micoscopy
- TEM (Transmission "")
- 60-90nm of tissue
- SEM (Scannning "")
- Interactions with the beam and the specimen generate
- Second electrons which are sent to a monitor
- tissue which has been coated with a heavy metal
- Interactions with the beam and the specimen generate
- E- are condensed and focused on a specimen
- TEM (Transmission "")
- Atomic Force Microscopy
- angstrom resolution
- silicon-based cantilever focused an infrared laser
- Described based on their energy source
- Artifacts
- Defects
- can occur in any stage
- Old or ill-prepared reagents
- impure chemicals
- Bad timing
- knife marks (chatter
- Light Microscopy
Tissue Processing
- Staining provides contrast to the samples
- IHC of frozen sections, secondary antibodies
- are conjugated to eitheran enzyme or a fluorophore
- TEM, heavy metals are used
- Osmium tetroxide
- both fixes and stains membranes
- uranyl acetate
- stains nuclear material
- lead
- stains proteins
- Osmium tetroxide
- Alternative fixatives to formaldehyde
- potassium permanganate
- osmium tetroxide
- acetone/methanol)
- acrolein (tear gas)
Data acquisition techniques
- Histochemistry and cytochemistry
- functional analysis of cells/tissues
- Enzyme histochemistry uses enzyme-substrate
- reactions to localize enzyme activity
- Immunohistochemistry
- uses primary antibodies directed against an epitope
- fluorescently-labeled secondary antibodies
- directed against the primary antibody
- May detect location of enzymes (not necessarily if they are active)
- fluorescently-labeled secondary antibodies
- Direct immunofluorescence employs a fluorescently-labeled primary antibody
- Limited Use
- useful in flow cytometry
- uses primary antibodies directed against an epitope
- Hybridization Techniques
- Uses the complementary base-pairing of a radiolabeled
- probe to a molecule of mRNA or DNA
- Can identify small numbers of nuclei acids
- (radio isotopes) 32P, 35S, 3H
- Uses the complementary base-pairing of a radiolabeled
- Autoradiography
- radiolabeled monomers
- Histochemistry and cytochemistry
Chemical Basis for Staining
- Basic dyes interact with anionic moieties
- phosphate groups
- carboxyl groups
- reaction of anionic groups varies with pH
- At a pH ~10; all three are sufficiently ionized to react with dye
- At pH 5-7; sulfate and phosphate groups are ionized
- At pH <4, only sulfate groups react
- sulfate groups
- Stains
- Hematoxylin
- Not a strict basic dye, similar properties
- used with an mordant
- Mordant makes it act as a basic dye
- used in sequential staining
- Eosin
- H&E Staining
- Loss of information is typical
- lipid extraction occurs
- Solvents are used for infiltration and curing
- The pH of the fixative may hydrolyze some molecules
- The aqueous nature may extract glycogen, proteoglycans, GAGs
- Loss of information is typical
- Acidic Dyes
- Eosin, acid fuchsin, aniline blue, orange G
- Net (-) charge on the dye
- react with cationic groups (acidophilia)
- via electrostatic interactions
- Basophilic substances
- heterochromatin
- nucleoli
- cytoplasmic components
- extracellular materials
- identify cytoplasmic filaments, intracellular membranous organelles, and extracellular fibers
- Basic dyes
- Methyl green, methylene blue, pyronin G, toluidine blue
- Net (+) charge on the dye
- Hematoxylin
- PAS stainscarbohydrate-rich macromolecules
- Glycogen
- mucus
- basement membrane
- Periodic acid-Schiff
- Schiff reagent
- Aldehyde groups and basic fuchsin interact, forms red color
- Periodic acid-Schiff (PAS) and Feulgen reactions take advantage of this interaction
- Feulgen reaction is based on cleavage of purines from deoxyribose
- Periodic acid-Schiff (PAS) and Feulgen reactions take advantage of this interaction
- distinct red color
- Aldehyde groups and basic fuchsin interact, forms red color
- Basic dyes interact with anionic moieties