Which compound functions as the substrate for HRP in typical IHC protocols?

• A. Naphthol-AS-phosphate
• B. Hydrogen peroxide
• C. DAB
• D. Tetramethylbenzidine

Answer: (B)

In HRP-based IHC, the enzyme needs an oxidant to drive the catalytic cycle and oxidize a chromogenic substrate to produce color. Hydrogen peroxide provides that oxidizing power, acting as the substrate for the HRP reaction. Without it, the enzyme cannot activate to oxidize the chromogen and generate the visible signal. DAB and TMB are chromogenic substrates that become colored when oxidized by HRP in the presence of hydrogen peroxide, but they are the substances being oxidized, whereas hydrogen peroxide is the actual substrate that enables the HRP reaction. Naphthol-AS-phosphate is used with alkaline phosphatase, not HRP.