AUTORADIOGRAPHIC VISUALIZATION OF S-35 LABELED CRNA PROBES COMBINED WITH IMMUNOPEROXIDASE DETECTION OF CHOLERAGENOID - A DOUBLE-LABELING LIGHT-MICROSCOPIC METHOD FOR IN-SITU HYBRIDIZATION AND RETROGRADE TRACT TRACING

We describe a protocol for simultaneous light microscopic visualizatio n of a neuron's efferent projections and its expression of mRNA. We ha ve combined immunohistochemical visualization of the retrograde marker cholera toxin subunit B (CTb) with autoradiographic visualization of S-35-labeled cRNA probes. Injections of CTb were made into rat brain. Immunoreactivity for CTb was demonstrated by modification of the perox idase-anti-peroxidase immunohistochemical technique, with DAB and nick el ammonium sulfate or cobalt acetate as chromogen. On the same sectio ns, in situ hybridization was performed with a S-35-labeled RNA probe complementary to preproenkephalin mRNA or tyrosine hydroxylase mRNA. M any double-labeled neurons were detected. These neurons contained pero xidase reaction product and were covered by an accumulation of silver grains in the overlaying emulsion layer. The present method has severa l advantages over double-labeling methods using the combination of flu orescent tracers and oligonucleotide probes. Both reaction products ar e permanent and can be visualized simultaneously by light microscopy. Furthermore, both CTb and cRNA probes are very sensitive markers. In a ddition, the sections can be counterstained.