In IHC, the binding of an antibody to a specimen depends on what condition?

• A. The pH of the buffer
• B. Time of day
• C. The instrument used
• D. The presence of the proper antigen

Answer: (D)

The key idea in this question is that antibody binding in IHC requires the target antigen to be present and accessible in the tissue. An antibody is designed to recognize a specific epitope on its antigen, so if the antigen isn’t expressed or the epitope is masked by fixation, there’s nothing for the antibody to bind to and no specific signal will appear, regardless of how good the antibody or protocol is. Factors like antigen retrieval can restore epitope accessibility after fixation, and staining conditions (buffer pH, incubation time, instrument) can influence signal strength and background, but they don’t create binding where the antigen isn’t present. Therefore, the presence and accessibility of the proper antigen is the fundamental condition for successful antibody binding in IHC.