Autoradiographic visualization of 35S-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 visualization of a neuron's efferent projections and its expression of mRNA. We have combined immunohistochemical visualization of the retrograde marker cholera toxin subunit B (CTb) with autoradiographic visualization of 35S-labeled cRNA probes. Injections of CTb were made into rat brain. Immunoreactivity for CTb was demonstrated by modification of the peroxidase-anti-peroxidase immunohistochemical technique, with DAB and nickel ammonium sulfate or cobalt acetate as chromogen. On the same sections, in situ hybridization was performed with a 35S-labeled RNA probe complementary to preproenkephalin mRNA or tyrosine hydroxylase mRNA. Many double-labeled neurons were detected. These neurons contained peroxidase reaction product and were covered by an accumulation of silver grains in the overlaying emulsion layer. The present method has several advantages over double-labeling methods using the combination of fluorescent tracers and oligonucleotide probes. Both reaction products are permanent and can be visualized simultaneously by light microscopy. Furthermore, both CTb and cRNA probes are very sensitive markers. In addition, the sections can be counterstained.