Diaminobenzidine: 60 years of use in the life sciences, and still going strong

Since the introduction of diaminobenzidine (DAB) by Richard Graham and Morris Karnovsky 60 years ago, it has enjoyed remarkable success in the life sciences. In this review, the chemistry, reactivity, and polymerization of DAB, and speculations around the coordination chemistry of its polymer, polyDAB, are discussed, and notable developments in the use of DAB in the life sciences, since a previous review in 1979, are described. Histochemical applications include the localization of hydrogen peroxide in plant tissues that accumulates as a consequence of pathology, the metal-catalyzed polymerization of DAB for visualizing endogenous metal complexes, for increasing the visibility of histochemical deposits such as Perl's stain, and for localizing metal oxide nanoparticles. In enzyme histochemistry, DAB is still used for demonstrating cytochrome oxidases activity, but its polymerization by exogenously applied horseradish peroxidase (HRP) is where it gained particular prominence both in neuronal tracing, and especially in immunohistochemistry. The physical inhibition of enzymatic activity, following deposition of polyDAB, that limited immunohistochemical sensitivity led to a variety of methods to address this issue. These exploited the ability of polyDAB to form complexes with metals, resulting in an increase in its intensity and sometimes also its color, its capacity to be amplified by silver-based development, or both. The alteration in the color of polyDAB was also employed for multiple sequential immunostaining with HRP or when immunohistochemistry was combined with other histochemical techniques. More recently, DAB has found utility in the ultrastructural localization of specific proteins that have been genetically labeled with peroxidase tags or fluorochromes, the latter being visualized by the process of photoconversion which exploits the fact that fluorescence excitation can be coupled to the polymerization of DAB. Finally, the future of DAB in the life sciences, whether in its native form or some modified version, is speculated on.