Inadequate Detection System Sensitivity (Immunohistochemistry (CST Guide))

Question

Accepted Answer

Weak signal or no staining despite proper primary antibody performance. Standard HRP-conjugated secondary antibodies fail to provide sufficient signal amplification. Common causes: 1) Using biotin-based detection systems which are less sensitive than polymer-based systems 2) Standard secondary antibodies directly conjugated with HRP lack sufficient signal amplification 3) Expired detection reagents used in protocol 4) Biotin-based detection used on tissues with high endogenous biotin (kidney, liver) causing interference Solutions: 1) Use polymer-based detection reagents (SignalStain Boost IHC Detection Reagents #8114 for rabbit, #8125 for mouse) instead of biotin-based systems 2) Combine polymer detection with SignalStain DAB Substrate Kit #8059 for enhanced sensitivity 3) Verify expiration date of all detection reagents prior to use 4) For kidney/liver tissues with high endogenous biotin, switch from biotin-based to polymer-based detection system