HT Immunohistochemistry Procedures 2 — Questions and Answers
Question 1: Which antigen retrieval method uses a pressure cooker or autoclave to expose epitopes?
- Enzymatic digestion
- Heat-induced epitope retrieval (HIER) (Correct answer)
- Microwave retrieval only
- Proteinase K digestion
Correct answer: Heat-induced epitope retrieval (HIER)
Heat-induced epitope retrieval (HIER) uses high-temperature methods such as pressure cookers, autoclaves, or water baths to unmask formalin-fixed epitopes.
Question 2: What is the purpose of a positive control slide in IHC?
- To test the specificity of the secondary antibody alone
- To verify that the entire IHC procedure is working correctly (Correct answer)
- To determine the optimal antibody dilution
- To check for nonspecific background staining
Correct answer: To verify that the entire IHC procedure is working correctly
A positive control slide contains tissue known to express the target antigen, confirming that the staining procedure, reagents, and antibodies are all functioning correctly.
Question 3: In a streptavidin-biotin IHC system, which molecule binds directly to the primary antibody?
- Streptavidin
- Chromogen
- Biotinylated secondary antibody (Correct answer)
- Enzyme conjugate
Correct answer: Biotinylated secondary antibody
The biotinylated secondary antibody binds to the primary antibody, after which streptavidin-enzyme complex binds the biotin tag.
Question 4: Which chromogen produces a brown end product when used with horseradish peroxidase in IHC?
- Fast Red
- DAB (3,3'-diaminobenzidine) (Correct answer)
- BCIP/NBT
- AEC
Correct answer: DAB (3,3'-diaminobenzidine)
DAB (3,3'-diaminobenzidine) reacts with horseradish peroxidase to produce a permanent brown precipitate at the antigen site.
Question 5: Endogenous peroxidase activity in tissue is typically blocked using:
- Normal goat serum
- Hydrogen peroxide in methanol or water (Correct answer)
- Avidin-biotin blocking solution
- Citrate buffer pH 6.0
Correct answer: Hydrogen peroxide in methanol or water
Hydrogen peroxide solution quenches endogenous peroxidase activity, preventing false-positive background when using HRP-based detection systems.
Question 6: Which IHC staining pattern is typical for Ki-67, a proliferation marker?
- Cytoplasmic granular
- Membranous
- Nuclear (Correct answer)
- Perinuclear dot-like
Correct answer: Nuclear
Ki-67 is a nuclear protein expressed during active cell proliferation, producing a nuclear staining pattern in IHC.
Question 7: When performing double IHC staining, which of the following is most important to prevent antibody cross-reactivity?
- Using the same species for both primary antibodies
- Using primary antibodies from different species (Correct answer)
- Applying both primary antibodies simultaneously
- Using the same chromogen for both targets
Correct answer: Using primary antibodies from different species
Using primary antibodies raised in different species (e.g., mouse and rabbit) allows species-specific secondary antibodies to distinguish between the two targets.
HT Immunohistochemistry Procedures 2
Which antigen retrieval method uses a pressure cooker or autoclave to expose epitopes?