IMMUNOHISTOCHEMISTRY (IHC) Flashcards
A technique for identifying ANTIGENS by means of ANTIGEN-ANTIBODY INTERACTIONS, the site of antibody binding being identified either by direct labeling of the antibody, or by use of a secondary labeling method
Immunohistochemistry
Primary antibody binds directly to:
UNKNOWN ANTIGEN
Secondary antibody binds to the:
PRIMARY ANTIBODY, often conjugated with a detection enzyme or fluorochrome
Most commonly used antibody for immunohistochemistry
IgG
Most frequently used animal to produce POLYCLONAL ANTIBODIES
RABBIT
Rabbit>Goat>Pig>Sheep>Horse>GUINEA PIG
Most frequently used animal to produce MONOCLONAL ANTIBODIES:
MICE
Fixative of choice for IHC
FORMALIN
Adhesive for IHC
POLY-L-LYSINE
Most commonly used antibody for IHC
IgG
Most commonly used animal for retrieving monoclonal antibodies
MICE
Most commonly used animal for retrieving polyclonal antibodies:
RABBIT
Most commonly used enzyme label for IHC
HORSE RADISH PEROXIDASE
A common fluorochrome used in IHC
FLUORESCENCE ISOTHIOCYANATE
Color of chromogen Diaminobenzidine (DAB)
BROWN
Color of chromogen Aminoethylcarbazole (AEC)
RED
Secondary/nuclear counterstain used in IHC
HEMATOXYLIN
In IHC, _____________ is raised against specific cellular _________and then conjugated with a __________
In IHC, ANTIBODY is raised against specific cellular ANTIGEN and than conjugated with a VISUAL MARKER
Best positive control for IHC
IDENTICAL TO PATIENT TISSUE
Size of paraffin sections for IHC
3-5 micra
Microscopes used in IHC
LIGHT OR FLUORESCENT
IMMUNOHISTOCHEMISTRY TECHNIQUES:
- Direct technique
- Indirect technique
- Peroxidase-antiperoxidase (PAP) technique
- Avidin-Biotin Complex (ABC) technique
- Labeled Streptavidin Avidin Biotin (LSAB) technique
- Immunofluorescence Methods
Traditional technique; Conjugate the PROIMARY ANTIBODY directly to the label such as fluorochrome or horseradish peroxidase
DIRECT TECHNIQUE
More sensitive than the traditional direct technique; TWO or THREE STEP PROCEDURE that involves application of unconjugated primary antibody, followed by a labeled antibody directed against the first antibody
INDIRECT TECHNIQUE
The most commonly used enzyme for indirect antibody enzyme-complex techniques
HORSERADISH PEROXIDASE (HRP)
An INDIRECT ANTIBODY ENZYME-COMPLEX TECHNIQUE where the soluble peroxidase-antiperoxidase complex is bound to unconjugated primary antibody by a second layer of “bridging” antibody, usually a swine anti-rabbit antibody, that then binds to both the primary antibody and the rabbit PAP complex
PEROXIDASE-ANTIPEROXIDASE (PAP) TECHNIQUE
In PAP, combining horseradish peroxidase with the most common chromogen ________________ results in a stable insoluble ____________________ end product when antigen is present in the tissue
In PAP, combining horseradish peroxidase with the most common chromogen DIAMINOBENZIDINE (DAB) results in a stable insoluble DARK BROWN REACTION end product when antigen is present in the tissue
REMEMBER!!!!
PAP is the peroxidase staining procedure in which reagents are link exclusively by antigen-antibody reactions WITHOUT ANY CONJUGATION STEPS
In Immunoperoxidase staining, efficiency of the chromogen-substrate reaction step can be checked by:
OMITTING THE BLOCKAGE OF ENDOGENOUS PEROXIDASE
Uses AVIDIN derived from egg white because of its marked affinity for BIOTIN, a low molecular weight vitamin that can be easily conjugated and enzyme markers
AVIDIN-BIOTIN COMPLEX (ABC) TECHNIEUQ
A method that is found to be 4 to 8 times MORE SENSITIVE than the old ABC method; an immunohistochemistry labelling method in which a molecule of avidin is directly labeled with an enzyme
LABELED STREPTAVIDIN AVIDIN BIOTIN (LSAB) Technique
The most common tissue processing method for fluorescence microscopy techniques is frozen section fixed in ice cold acetone
IMMUNOFLUORESCENCE METHODS
Tissues most often test for diagnostics using fluorescence-labeled-antibodies:
KIDNEY and SKIN
For solid tissue biopsies; tissue is reacted with FLUORESCEIN-CONJUGATED ANTIBODY specific for the material being sought within the tissue
DIRECT IMMUNOFLUORESCENCE
Used for the detection of AUTOANTIBODIES in the patient’s serum including anti-nuclear antibody (ANA), anti-mitochondrial antibody (AMA), and liver-kidney microsomal antibody
INDIRECT IMMUNOFLUORESCENCE
Based on the specificity of the interaction of a probe with the target NUCLEIC ACIDS; The detection procedure for an in-situ hybridization probe is SIMILAR to IHC. However it is based on the specificity of the interaction of a probe with the target NUCLEIC ACID, rather than the target protein or immunogen
IN-SITU HYBRIDIZATION
