A New Specific, Sensitive And Non-carcinogenic Reagent For The Demonstration Of Horseradish Peroxidase

3,3'-Diaminobenzidine (DAB), introduced by Graham & Karnovsky (J. Histochem. Cytochem. 1966, 14, 291 & J. exp. Med. 1966, 124, 1123) is one of the most widely employed reagents in histochemistry. It has been very useful in demonstrating the sites to which the exogenous ultrastructural tracer horseradish peroxidase (HRP) is transported in vertebrate tissues. Over the past five years, DAB staining of transported HRP in neurons has developed into a powerful neuroanatomical tool for labelling the cells of origin of pathways in the central nervous system (La Vail, 1975, in: The use o f axonal transport for studies o f neuronal connectivity (eds. W. M. Cowan & M. Cuenod), pp. 217-248. Amsterdam: Elsevier.). Reports of the borderline carcinogenicity of DAB in rats (Griswold, Casey, Weisburger & Weisburger, Cancer Res. 1968, 28,924) may be responsible for the decreased availability of good quality DAB. Recent studies (Hanker, Anderson & Bloom, Science 1972, 175, 991; Hanker & Rabin, J. clin. Mierobiol. 1975, 2, 463) suggested that oxidative coupling reactions of aromatic amines in the presence of phenols might provide a suitable substitute for DAB. Some of these reactions yield deeply-coloured synthetic melanin-like compounds which are osmiophilic and sufficiently insoluble to be suitable end-products for histochemistry. In addition, the reaction must be sufficiently rapid to deposit the end-product at the sites of the plant hydroperoxidase alone. Such a reaction was realized when it was found that the peroxidation of p-phenylenediamine (PPD) was greatly accelerated by the presence of pyrocatechol (PC). The co-polymer formed as a result of this oxidative coupling reaction was osmiophilic and was bluer than oxidized DAB. It was insoluble and conformed well to biological ultrastructure. Neither PPD nor PC used individually gave satisfactory results and the best results were obtained when a mixture of 1 part (by weight) PPD to 2 parts PC was employed as the reagent. The administration of HRP and fixation of tissue for studies in mice, other than those involving axonal transport, were carried out according to Graham & Karnovsky (J. Histochem. Cytochem. 1966, 14, 291 &J. exp. Med. 1966, 124, 1123). For the demonstration of retrograde axonal transport in cats, rats, and rhesus monkeys, 30% HRP (Boehringer) in distilled water was injected into the sensorimotor cortex, or the ventrobasal complex of the thalamus (VB injection) or in the cerebellar cortex. The volume of HRP solution injected varied from 0.05 to 2/al. The animals were perfused with a mixture of 0.5% (para)formaldehyde and 2.5% glutaraldehyde in 0. t M phosphate buffer, pH 7.2. Tissue blocks (brain) were excised and immersed in 30% sucrose in 0.1 M phosphate buffer (pH 7.2) immediately after the perfusion. Cryostat or Vibratome* sections of mouse tissues were cut at 10-20/~m. Frozen sections of the rat, cat or monkey brains were cut at 40/2m.