Why is it necessary to quench endogenous peroxidase when using DAB?

• A. It prevents nonspecific binding of antibodies.
• B. DAB is oxidized by hydrogen peroxide in the presence of Hemoglobin giving blood cells a dark brown color.
• C. It preserves tissue morphology.
• D. It enhances antigen retrieval.

Answer: (B)

DAB needs both peroxidase and hydrogen peroxide to form the brown precipitate that marks where the antibody is bound. Tissues contain endogenous peroxidase activity, especially in blood-rich areas, which can react with DAB in the presence of H2O2 and produce brown staining independent of the target antigen. Quenching endogenous peroxidase blocks this background reaction, so the brown signal reflects only the specific antibody-antigen interaction instead of nonspecific enzyme activity. Other options describe aspects like blocking nonspecific binding, preserving tissue morphology, or unmasking epitopes, which don’t address the issue of background from endogenous peroxidase.