False-Positive Signal from Endogenous Enzyme Activity

Symptom

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.

1. 1 Endogenous alkaline phosphatase activity with AP-conjugated antibodies
2. 2 Endogenous peroxidase activity with HRP-conjugated antibodies
3. 3 Lack of enzyme inhibitor treatment prior to immunostaining
4. 4 Insufficient endogenous enzyme blocking step

1. 1 Block endogenous alkaline phosphatase with 2 mM Levamisol (ab141217) for AP-conjugated antibodies
2. 2 Block endogenous peroxidase with 0.3% (v/v) H2O2 for HRP-conjugated antibodies
3. 3 For fragile samples requiring morphology preservation, dilute hydrogen peroxide in methanol
4. 4 For cell surface or membrane proteins, use hydrogen peroxide in PBS or quench after primary antibody incubation

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