Inadequate Detection System Sensitivity

Symptom

Weak signal or no staining despite proper primary antibody performance. Standard HRP-conjugated secondary antibodies fail to provide sufficient signal amplification.

1. 1 Using biotin-based detection systems which are less sensitive than polymer-based systems
2. 2 Standard secondary antibodies directly conjugated with HRP lack sufficient signal amplification
3. 3 Expired detection reagents used in protocol
4. 4 Biotin-based detection used on tissues with high endogenous biotin (kidney, liver) causing interference

1. 1 Use polymer-based detection reagents (SignalStain Boost IHC Detection Reagents #8114 for rabbit, #8125 for mouse) instead of biotin-based systems
2. 2 Combine polymer detection with SignalStain DAB Substrate Kit #8059 for enhanced sensitivity
3. 3 Verify expiration date of all detection reagents prior to use
4. 4 For kidney/liver tissues with high endogenous biotin, switch from biotin-based to polymer-based detection system

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