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Blocking Endogenous Peroxidase Activity

Endogenous peroxidase blocking is a necessary step in methods that use a peroxidase-based detection system. If this step is not performed, cells that have endogenous peroxidase (RBCs) will appear as positive. The most common method is a 5 to 10 minute incubation with 0.3% to 3.0% hydrogen peroxide (H2O2) in phosphate buffered saline (PBS), methanol, or distilled water at room temperature. This procedure may be performed before the application of the primary antibody or after the link antibody incubation. Using the methanol based H2O2 solution is harsher than the other mixtures. This is commonly used with blood smears, bloody/inflamed, and/or vascular tissues. There are pre-mixed solutions available that not only block endogenous peroxidase activity but some of the other unwanted enzymatic activity as well.