HT Immunohistochemistry Procedures 3 — Questions and Answers
Question 1: What is the main advantage of polymer-based IHC detection systems over avidin-biotin systems?
- Lower cost per test
- Elimination of endogenous biotin interference (Correct answer)
- Faster chromogen development
- Stronger nuclear counterstaining
Correct answer: Elimination of endogenous biotin interference
Polymer-based systems avoid biotin entirely, eliminating false positives caused by endogenous biotin found in liver, kidney, and other tissues.
Question 2: Which tissue type is most likely to cause false-positive results due to high endogenous biotin content?
- Skeletal muscle
- Liver and kidney (Correct answer)
- Squamous epithelium
- Peripheral nerve
Correct answer: Liver and kidney
Liver and kidney contain high levels of endogenous biotin, which can bind streptavidin and cause background staining in avidin-biotin-based IHC systems.
Question 3: Which pH is most commonly used with citrate buffer for heat-induced antigen retrieval?
- pH 4.5
- pH 6.0 (Correct answer)
- pH 7.4
- pH 9.0
Correct answer: pH 6.0
Citrate buffer at pH 6.0 is the most widely used HIER solution for unmasking antigens fixed with formalin in routine histopathology.
Question 4: Which IHC marker is commonly used to identify T-lymphocytes in tissue sections?
- CD20
- CD3 (Correct answer)
- CD138
- CD68
Correct answer: CD3
CD3 is a pan-T-cell marker expressed on the surface of T-lymphocytes and is routinely used in IHC to identify T-cells in tissue.
Question 5: In IHC, the purpose of a blocking step with normal serum is to:
- Enhance primary antibody binding
- Reduce nonspecific background by blocking free tissue protein binding sites (Correct answer)
- Activate the secondary antibody
- Prevent chromogen precipitation
Correct answer: Reduce nonspecific background by blocking free tissue protein binding sites
Normal serum from the same species as the secondary antibody saturates nonspecific binding sites in the tissue, reducing background staining.
Question 6: Which counterstain is most commonly used with DAB chromogen in IHC?
- Eosin
- Hematoxylin (Correct answer)
- Methylene blue
- Safranin O
Correct answer: Hematoxylin
Hematoxylin provides a blue nuclear counterstain that contrasts with the brown DAB signal, allowing morphological assessment alongside IHC results.
Question 7: A negative control in IHC is prepared by:
- Omitting the primary antibody and replacing with diluent (Correct answer)
- Using a different chromogen
- Omitting the secondary antibody only
- Increasing the antigen retrieval time
Correct answer: Omitting the primary antibody and replacing with diluent
The negative control omits the primary antibody (replaced with antibody diluent) to confirm that any staining observed in the test section comes from the primary antibody's specific binding.
HT Immunohistochemistry Procedures 3
What is the main advantage of polymer-based IHC detection systems over avidin-biotin systems?