ASCP Immunohistochemistry 1 — Questions and Answers
Question 1: What is the purpose of antigen retrieval in immunohistochemistry (IHC)?
- To deparaffinize sections
- To unmask epitopes altered by formalin fixation (Correct answer)
- To block endogenous peroxidase
- To apply the secondary antibody
Correct answer: To unmask epitopes altered by formalin fixation
Formalin cross-links proteins and masks antigenic epitopes; antigen retrieval (heat or enzyme-based) reverses these cross-links to restore antibody binding.
Question 2: Which of the following describes the avidin-biotin complex (ABC) method in IHC?
- Direct antibody-chromogen system
- Indirect amplification system using avidin-biotin affinity (Correct answer)
- Enzyme-only detection system
- Fluorescent antibody method
Correct answer: Indirect amplification system using avidin-biotin affinity
The ABC method uses a biotinylated secondary antibody that binds to a preformed avidin-biotin-enzyme complex, amplifying signal through the high-affinity avidin-biotin interaction.
Question 3: Why is endogenous peroxidase activity blocked in IHC protocols using HRP-based detection?
- To prevent tissue damage
- To eliminate background staining from naturally occurring peroxidases (e.g., in RBCs) (Correct answer)
- To improve antigen retrieval
- To prevent antibody aggregation
Correct answer: To eliminate background staining from naturally occurring peroxidases (e.g., in RBCs)
Endogenous peroxidases in hemoglobin and other cellular components will react with the DAB chromogen, creating false-positive background staining if not blocked.
Question 4: What chromogen is most commonly used with HRP-based IHC and produces a brown reaction product?
- BCIP/NBT (blue)
- DAB (3,3'-diaminobenzidine) (Correct answer)
- AEC (red)
- Fast Red
Correct answer: DAB (3,3'-diaminobenzidine)
DAB (3,3'-diaminobenzidine) is oxidized by HRP in the presence of hydrogen peroxide to produce an insoluble brown precipitate at antigen sites.
Question 5: What is a positive control in IHC and why is it required?
- A tissue section without primary antibody to check background
- A tissue known to contain the target antigen, run to confirm the staining protocol works (Correct answer)
- A section stained only with the chromogen
- A tissue treated with buffer instead of antibody
Correct answer: A tissue known to contain the target antigen, run to confirm the staining protocol works
A positive control (tissue with known antigen expression) confirms that antibody, detection reagents, and staining conditions are functioning correctly.
Question 6: Which heat-based antigen retrieval method is most commonly used in routine IHC?
- Microwave in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) (Correct answer)
- Autoclave in distilled water
- Water bath in ethanol
- Pressure cooker in acetone
Correct answer: Microwave in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)
Heat-induced epitope retrieval (HIER) using citrate buffer at pH 6 or EDTA at pH 9 in a microwave or steamer is the standard method for most antibodies.
ASCP Immunohistochemistry 1
What is the purpose of antigen retrieval in immunohistochemistry (IHC)?