What does it indicate when a staining reaction is observed despite using a blocking peptide in an IHC protocol?

• A. Specific staining
• B. Non-specific staining or false positive staining
• C. Tissue degradation
• D. Complete absence of signal

Answer: (B)

The blocking peptide is used to test whether the antibody’s binding is truly specific to the target antigen. If staining is still seen after pre-absorption with the blocking peptide, it means the signal is not due to the specific antigen–antibody interaction but to non-specific binding or cross-reactivity. In other words, it’s a false positive: the antibody or detection system is producing a signal through other interactions (such as binding to other proteins, Fc receptors, secondary antibody cross-reactivity, or endogenous enzymes) rather than the intended target. This contrasts with true specific staining, which should be eliminated or greatly diminished when the antibody is blocked.