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Immunohistochemistry Flashcards

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

    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.

  2. Which of the following describes the avidin-biotin complex (ABC) method in IHC?

    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.

  3. Why is endogenous peroxidase activity blocked in IHC protocols using HRP-based detection?

    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.

  4. What chromogen is most commonly used with HRP-based IHC and produces a brown reaction product?

    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.

  5. What is a positive control in IHC and why is it required?

    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.

  6. Which heat-based antigen retrieval method is most commonly used in routine IHC?

    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.