Osmiophilic reagents in electronmicroscopic histocytochemistry.
Direct histocytochemical staining methods on undisrupted tissues, stabilized by chemical fixation, potentially offer perhaps the most reliable approach to the study of the enzymes of the cell with relation to its ultrastructure. The atoms which, for the most part, comprise the biomacromolecules and enzymes of cells and tissues contribute little to their inherent electron opacity or ability to scatter electrons differentially. The latter property of a substance is responsible for its observation with the electron microscope. Since the introduction of osmiophilic reagents into cytochemistry (HANKER et al. 1964), the selective deposition of relatively large amounts of polymeric osmium black reaction products at the subcellular sites of insoluble or immobilized enzymes or biomacromolecules has facilitated their demonstration with the light and electron microscopes. Perhaps the most widely employed osmiophilic reagent in histocytochemistry has been DAB which was introduced by GRAHAM and KARNOVSKY (1966a, b). Although it receives its widest use for demonstrating the sites to which the exogenous ultrastructural tracer horseradish peroxidase (HRP) is transported in vertebrate tissues, it is also widely employed for the demonstration of catalase in peroxisomes with the media of FAHIMI (1969) or of NOVIKOFF and GOLDFISCHER (1969), and for the demonstration of cytochrome oxidase with the medium of SELIGMAN et al. (1968a). The importance of this reagent lies in its ability to undergo oxidative polymerization forming an insoluble osmiophilic melanin-like product (HANKER et al. 1972a) which comforms well to ultrastructure, at the sites of enzymic or nonenzyme proteins which catalyze its oxidation. In the past few years, studies in our laboratory have shown that a rational approach to the histocytochemical demonstration of enzymes could be devised. It is based on the selective deposition of transition metal compounds at the sites of enzymes that resemble hemoproteins in their ability to catalyze the oxidative polymerization of DAB. The most useful of these compounds, cupric ferrocyanide (Hatchett's brown) was also introduced into cytochemistry by Karnovsky's laboratory (KARNOVSKY 1964; KARNOVSKY and ROOTS 1974). By the use of natural substrates, when available, or synthetic substrates which liberate or form a reducing agent at the sites of the enzymatic activity, many diverse types of enzymes have been demonstrated by methods depending on this principle known as catalytic osmiophilic polymer generation. DAB has probably been the most useful histocytochemical reagent of the past decade. Yet its borderline carcinogenicity and the frequent interruption of a supply of good quality DAB have encouraged research into a substitute reagent. A new substitute for DAB has resulted from the study of artificial melanins in our laboratory for several years. It consists of a mixture of p-phenylenediamine and pyrocatechol and is much better than DAB for the demonstration of HRP used as a cytochemical tracer...