IMMUNOHISTOCHEMISTRY Flashcards
Identification of specific tissue or cellular antigens, found on tissues
Immunohistochemistry
Used for detection of organisms in cytologic preparations (fluids, sputum, FNA)
Immunohistochemistry Application
Most commonly used antibody class
IgG
Produced by different cells, reacts with various epitopes, common source rabbit
Polyclonal Antibodies
Other animal sources for polyclonal antibodies
Goat, pig, sheep, horse, guinea pig
Source of monoclonal antibodies
Mice
Storage of aliquots in immunohistochemistry
-80°C
Enzyme labels in Immunohistochemistry
HRP (Horse Radish Peroxidase) and AP/ALP (Alkaline Phosphatase)
Common chromogens
DAB (Diaminobenzidine), AEC (brick red), Nitroblue tetrazolium, Fast Red TR salt
Preferred chromogen for permanent stains
DAB
Chromogen for skin sections
AP Red (to prevent melanin masking)
Double staining method
DAB and AP in the same section
Incubation time for antibody-peroxidase linkage
30-60 minutes at room temp
End product visualization
Light microscopy
Fluorochrome used in IHC
FITC (Fluorescein Isothiocyanate)
Requirement for fluorochrome detection
Fluorescent microscope
Lectins definition
Plant/animal proteins binding to tissue CHO, can be labeled like antibodies
Fixation for frozen sections
Absolute methanol or acetone
Fixation for paraffin embedded tissue
10% Neutral Buffered Formalin
Other fixatives used
4% paraformaldehyde, Bouin’s, Zamboni’s
Unmasking requirement
For paraffin embedded tissues before IHC
Common counterstains
Hematoxylin, Methyl green, Nuclear fast red
Adhesive for tissue sections
APES diluted in acetone
Inactivators for blocking enzymes
3% Hydrogen Peroxide for HRP, Levamisole for ALP
Blocking agents purpose
Animal serum to block non-specific binding
Blocking time
10-30 minutes at room temperature
Mounting medium requirement
Neutral pH, 50% glycerol for short-term storage
DAB; Counterstain: Hematoxylin, Methyl Green, Methyl Blue; Mounting Medium: Neutral
HRP with DAB
AEC; Counterstain: Hematoxylin, Methyl Blue; Mounting Medium: Water Soluble
HRP with AEC
BCIP/NBT; Counterstain: Nuclear Fast Red, Brilliant Green; Mounting Medium: Neutral
AP with BCIP/NBT
Fast Red; Counterstain: Hematoxylin, Methyl Green, Brilliant Green; Mounting Medium: Water Soluble
AP with Fast Red
Tissue preparation
Deparaffinization > Inactivation using 3% hydrogen peroxide > Antigen retrieval > Blocking > Application of antibody > Staining > Mounting
A section known and proven to have the antigen in question
Positive control
Omit the primary antibody from the staining schedule. Replace the specific primary antibody with an Ig directed against an unrelated antigen
Negative control
Contains the target antigen but not in the tissue elements under investigation
Internal tissue control
Extended Polymer One Step Staining; direct labeling system with primary Ab conjugated to labeled long-chain dextran polymer
EPOS
More sensitive than direct; increases signal amplification using labeled secondary Ab
Two-step indirect technique
Secondary Ab conjugated to enzyme-labeled polymer chain
Polymer Chain Two-step indirect technique
