Tips for Step 6: Block Endogenous Enzymes and Biotin

To Avoid Staining Artifacts it is Important

To block endogenous peroxidases and phosphatases prior to using AP/horseradish peroxidase (HRP) antibody conjugates
For blocking of endogenous peroxidase activity, use hydrogen peroxide solutions such as Bio-Rad’s ready-to-use Peroxide Blocking Reagent (BUF017B; 3% (v/v) hydrogen peroxide). However, for the detection of certain antigens, such as CD4, this percentage of hydrogen peroxide may be too harsh (Kim et al. 2016)
To block endogenous biotin when using avidin/biotin or streptavidin/biotin detection systems. For this specific purpose Bio-Rad offers a ready-to-use Avidin/Biotin Blocking Reagent (BUF016)
To block endogenous AP activity supplement the substrate solution with levamisole (Ponder and Wilkinson 1981). According to Ponder and Wilkinson (1981), 20% acetic acid may also be used. However, this method may not be suitable for all antigens (Ponder and Wilkinson 1981)

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