A new double labeling technique using tyrosine hydroxylase and dopamine-beta-hydroxylase immunohistochemistry: evidence for dopaminergic cells lying in the pons of the beef brain.

Despite numerous data on the organization of the central catecholaminergic (CA) system, the nature of many CA terminal fields, fiber pathways and cell bodies, remains unknown. The discrepancies between the results obtained by CA histofluorescence and dopamine-beta-hydroxylase (DBH) immunofluorescence method suggest that certain areas described as noradrenergic (NA) may be in fact dopaminergic (DA). A method to solve this problem is to compare the localization of the two CA synthetizing enzymes: tyrosine hydroxylase (TH) and DBH. The first enzyme is a marker of all CA neurons, whereas the second one is a specific marker of noradrenergic and adrenergic neurons. We decided to develop a technique which allows visualization of both antigens on a same section. We chose to combine immunofluorescence and immunoperoxidase techniques which yield very different staining. The optimal conditions for this double labeling technique were developed in bovine brain, the homologous system for our antibodies. The results obtained by this procedure provide evidence that DA cells together with NA cells extend throughout the rostral part of the pons, and outline a successful technique for a more extensive investigation of the DA system. The same method should be applicable to other investigations involving other antigens.