Insufficient Detection System Sensitivity (Immunohistochemistry (CST Guide))

Question

Accepted Answer

Signal is weak or undetectable despite correct antibody and protocol, particularly for low-abundance targets, indicating that the detection system lacks sufficient signal amplification. Common causes: 1) Using biotin-based detection system instead of more sensitive polymer-based system 2) Using standard HRP-directly-conjugated secondary antibodies that provide insufficient amplification 3) Detection reagents (HRP or DAB substrate) expired or improperly stored Solutions: 1) Switch to polymer-based detection (SignalStain® Boost IHC Detection Reagent #8114 for rabbit primary, #8125 for mouse primary) for enhanced sensitivity 2) Use SignalStain® DAB Substrate Kit (#8059) in conjunction with polymer-based detection reagents 3) Verify expiration dates on all detection reagents; replace if expired or stored improperly 4) Compare biotin-based vs. polymer-based detection side-by-side to confirm performance enhancement