COMBINATION OF IMMUNOCYTOCHEMISTRY AND INSITU HYBRIDIZATION IN THE SAME SEMITHIN SECTIONS - DETECTION OF MET-ENKEPHALIN AND PROENKEPHALIN MESSENGER-RNA IN THE HYPOTHALAMIC MAGNOCELLULAR DORSAL NUCLEUS OF THE GUINEA-PIG

We describe a procedure for combining pre-embedding peroxidase immunoc ytochemistry with pre-embedding autoradiographic in situ hybridization in the same vibratome sections of paraformaldehyde-fixed brain tissue . The simultaneous detection of Met-enkephalin (Met-enk)-immunoreactiv e product and pro-enkephalin (PE) mRNA in neurons of the magnocellular dorsal nucleus (MDN) in the guinea pig hypothalamus was carried out a s a model for this procedure. Vibratome slices were processed for Met- enk immunodetection followed by the incubation with a 45-base syntheti c oligonucleotide complementary to PE mRNA labeled with S-35. Tissues were embedded in araldite, cut into semi-thin sections, and processed for autoradiography. Many neurons double labeled for Met-enk and PE mR NA were viewed in the MDN. The histological quality and the spatial re solution of both signals were optimized, since precise intracellular l ocalization of hybridization sites was possible. This method allows si multaneous study of peptide immunoreactivity and mRNA expression level s in neurons within the same semi-thin sections. It may be useful for a variety of quantitative analyses, and might also be extended to ultr astructural analysis.