- 3,3′-Diaminobenzidine (DAB) is a chromogenic substrate widely used in immunohistochemistry (IHC), immunocytochemistry (ICC), and blotting techniques for the visualization of antigens. It is oxidized in the presence of hydrogen peroxide (H₂O₂) by enzymes such as horseradish peroxidase (HRP), producing an insoluble brown precipitate at the site of the enzymatic reaction. This localized deposition marks the position of the antigen–antibody complex in tissue or cell preparations, making DAB one of the most important and reliable substrates in histopathology and research.
- The appeal of DAB lies in its clarity, stability, and permanence. The brown precipitate formed during the reaction is insoluble in alcohol and xylene, allowing slides to be dehydrated, cleared, and permanently mounted for long-term storage. This permanence is especially valuable in clinical diagnostics, where archived slides must remain interpretable for years. In addition, the brown staining contrasts well with common nuclear counterstains, such as hematoxylin, providing clear visualization of tissue morphology alongside antigen localization.
- DAB is considered more sensitive and stable compared to other chromogens like 3-amino-9-ethylcarbazole (AEC) or 4-chloro-1-naphthol. It provides crisp signals without significant diffusion, ensuring precise localization of target antigens. Its stability also makes it compatible with digital pathology workflows, as DAB-stained slides can be scanned and stored without fading. These properties have made it the gold standard chromogen for HRP-based detection in histology and immunoassays.
- Despite its advantages, DAB has some safety considerations. It is classified as a suspected carcinogen and must be handled with caution in the laboratory, using protective equipment and proper waste disposal. Additionally, overdevelopment of DAB staining can obscure tissue details or lead to high background, so reaction times need to be carefully optimized.
