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20770962
Histology (Ch1 Methods)
Descrição
Histology
Sem etiquetas
histology
Mapa Mental por
Kc Y
, atualizado more than 1 year ago
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Criado por
Kc Y
mais de 4 anos atrás
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Resumo de Recurso
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
E- are condensed and focused on a specimen
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
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
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)
Direct immunofluorescence employs a fluorescently-labeled primary antibody
Limited Use
useful in flow cytometry
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
Autoradiography
radiolabeled monomers
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
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
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
distinct red color
Anexos de mídia
Microscopess (binary/octet-stream)
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