Over-Amplified Signal with Biotin-Based Detection Systems (Immunohistochemistry (High Background))

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Accepted Answer

Excessive background staining when using amplification techniques or biotin-based detection, either from high signal amplification or endogenous biotin binding to streptavidin/avidin. Common causes: 1) Excessive biotin conjugation to secondary antibody 2) Over-amplification of detection signal 3) Endogenous biotin in tissue binding to streptavidin or avidin 4) Too much substrate or prolonged substrate incubation time Solutions: 1) Reduce the amount of signal amplification (e.g., conjugate less biotin to secondary antibody) 2) Use Avidin/Biotin Blocking Kit as an additional blocking step before detection 3) Dilute the substrate to reduce signal intensity 4) Reduce substrate incubation time to minimize background development