IHC Flashcards
(35 cards)
Immunofluorescence (IF)
Visualization of antigens w/in cells using antibodies as fluorescent probes.
FITC color
emits green
TRITC color
emits red-orange
DAB color
brown
AEC color
red
Fluorochrome
Molecules that absorb and emit light
Two types of IF
Direct
Indirect
Direct
Used less frequently
Primary antibody conjugated to fluorescent dye
shorter staining time
Needs specificity
Higher cost
Less flexibility
Indirect
Primary antibody is unlabeled
Secondary antibody is conjugated w/dye
Greater sensitivity
Greater amplification
Cheaper
More colors
Cross reactivity potential
Need different species for secondary
Background staining increases
Photon
Colored light
Emitted when the excited electron falls back to original energy level
Excitation peak
Optimal wave length of light that produces the most fluorescence
Ideal fluorophore properties
Absorption peak & excitation peak available on the fluorescent microscope
Bright fluorescence
Narrow emission spectrum
Good photostability
Fluorescence overlap
Two or more probes used on the same sample, the emission spectrum can overlap
Applications of IF in clinical pathology
Analysis of antigens in frozen or fixed tissue
Observation of bacterial or parasitic specimens
Detection and localization of the presence of absence of DNA sequences
Two stains for Muscular Dystrophy
ATPase
NADH
Other fluorescent dyes
Thioflavin T
Thioflavin S
Nanometer range for visible light spectrum
400-700
Transmission Electron Microscope (TEM)
Transmits or deflects electrons
Produces 2D image
Black & white
Useful in muscle & kidney disease DX
Scanning Electron Microscope (SEM)
3D image
Release secondary electrons as it scans
Used primarily in research
Useful in studying cell surfaces (membranes)
Microns for IHC
3-4µ
Alkaline Phosphatase (AP)
Varies from fast red to fast blue
Proteolytic enzymes
Proteinase K
Trypsin
Pepsin
Protease
Pronase
Common buffer for AR
Citrate buffer pH 6.0
Tris-HCl pH 10
Tris-EDTA pH 8
AVOID drying in high temp ovens above 60°C
