Immunohistochemical localization of a phosphoprotein (B-50) isolated from rat brain synaptosomal plasma membranes.

The endogenous phosphorylation in vitro of at least 5 protein bands of rat brain synaptosomal membranes (SPM) is inhibited by the behaviorally active peptide ACTH1-24. One of these proteins, the phosphoprotein band B-50, was isolated from rat brain SPM by SDS-slab gel electrophoresis. An antiserum to B-50 was raised in rabbits. The presence of antibodies to B-50 in the antiserum was demonstrated by immunoperoxidase staining of cryostat sections of a polyacrylamide gel containing the antigen. The production of antibodies was monitored by an indirect immunofluorescence technique using cryostat sections of quick-frozen rat cerebellum. Immunofluorescent staining of the molecular and granular layers was observed, whereas the white matter and the perikarya and Purkinje cells of the cerebellum were not stained. With the use of the peroxidase-antiperoxidase (PAP) method, the immunohistochemical localization of the antigen in the molecular and granular layer of the cerebellum was confirmed. Regions rich in neuropil, like the CA1, CA2, CA3, CA4 and dentate gyrus showed an intense immunostaining. Thus, in agreement with the synaptic origin of B-50, the antiserum reacted with tissue components present in brian regions in synaptic contacts.