ASCP Immunohistochemistry

Question 1

What is the purpose of antigen retrieval in immunohistochemistry (IHC)?

Accepted Answer

To unmask epitopes altered by formalin fixation

Answer

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?

Accepted Answer

Indirect amplification system using avidin-biotin affinity

Answer

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?

Accepted Answer

To eliminate background staining from naturally occurring peroxidases (e.g., in RBCs)

Answer

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?

Accepted Answer

DAB (3,3'-diaminobenzidine)

Answer

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?

Accepted Answer

A tissue known to contain the target antigen, run to confirm the staining protocol works

Answer

A positive control (tissue with known antigen expression) confirms that antibody, detection reagents, and staining conditions are functioning correctly.

ASCP - Histotechnician Test Practice Test

ASCP - Histotechnician Test Practice Test

ASCP Immunohistochemistry