False-Positive Signal from Endogenous Enzyme Activity (Immunohistochemistry (High Background))

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Accepted Answer

High background signal observed when using enzyme-conjugated antibodies (HRP or AP), caused by endogenous alkaline phosphatase or peroxidase activity in tissue generating false-positive results. Common causes: 1) Endogenous alkaline phosphatase activity with AP-conjugated antibodies 2) Endogenous peroxidase activity with HRP-conjugated antibodies 3) Lack of enzyme inhibitor treatment prior to immunostaining 4) Insufficient endogenous enzyme blocking step Solutions: 1) Block endogenous alkaline phosphatase with 2 mM Levamisol (ab141217) for AP-conjugated antibodies 2) Block endogenous peroxidase with 0.3% (v/v) H2O2 for HRP-conjugated antibodies 3) For fragile samples requiring morphology preservation, dilute hydrogen peroxide in methanol 4) For cell surface or membrane proteins, use hydrogen peroxide in PBS or quench after primary antibody incubation