The reagent 3,3'-Diaminobenzidine (DAB) is a sensitive alternative to protein stains to enhance fingermarks in blood. In contact with blood, DAB is oxidized to give a brown solid precipitating on the bloody fingermark. Blood marks are first fixed with a solution of 5-sulfosalicylic acid. A buffered DAB and hydrogen peroxide solution is then applied, and the reaction is allowed to proceed over ～4 min. The reaction between hydrogen peroxide and DAB, catalyzed by the peroxidase activity of blood, gives a dark brown insoluble product. Dark brown enhanced marks, therefore, result. The treatment gives little background colouration, even on porous items, and no destaining procedure is required[1].

Figure. HRP-DAB-mediated immunolabeling of PdgfRα in paraffin-sectioned WAT. Black arrows indicate PdgfRα + cells marked by deposition of oxidized DAB, which is brown in color.

On dark, coloured and patterned backgrounds, the dark brown product can be difficult to visualize; however, the contrast may be increased by further development using a different coloured protein staining method. The colour of the DAB precipitate may also be enhanced by adding cobalt, copper or nickel to the DAB working solution. We note that this dye is carcinogenic, and spraying is discouraged. In addition, due to its toxicity, removing all excess solutions after optimal development is prudent. Solutions for DAB should be freshly prepared as the dye is somewhat unstable in the solution. Aqueous DAB solutions may be frozen until just before use to increase their shelf life.

Note: 3,3'-Diaminobenzidine (DAB) is a potential carcinogen. Wear gloves while handling it. After the reaction, DAB should be disposed of carefully. We deactivate DAB with chlorine bleach and bleach all glassware and disposable items that have come in contact with it.