A COMBINED IN-SITU HYBRIDIZATION AND IMMUNOFLUORESCENCE PROCEDURE ALLOWING VISUALIZATION OF PEPTIDE MESSENGER-RNA AND SEROTONIN IN SINGLE SECTIONS

We describe a novel procedure for combining immunocytochemistry with i n situ hybridisation. In contrast to previously published procedures, the technique involves immunofluorescence followed by in situ hybridiz ation and is particularly suitable for antigens which are labile or se nsitive to in situ hybridization processing. We have evaluated the tec hnique using 5-hydroxytryptamine (5-HT, serotonin) immunofluorescence and neuropeptide in situ hybridization employing S-35-labelled oligonu cleotide probes. Successful double labelling was obtained and showed t hat galanin messenger RNA (mRNA) is expressed by 5-HT immunoreactive c ells in the dorsal raphe nucleus of the rat. In contrast, somatostatin mRNA in the same region is expressed by a separate non-serotonergic c ell population. Double-labelled preparations produced using this techn ique can be conveniently viewed using epipolarised combined with epifl uorescent illumination. Careful analysis of procedural variables revea led that it is not possible to carry out high-sensitivity 5-HT immunoc ytochemistry following in situ hybridization. The immunostaining is mu ch poorer on slide-mounted sections than on free-floating sections, an d 5-HT appears to be lost during the in situ hybridization steps of de hydration/delipidation and incubation in hybridization buffer. The pro cedure we describe avoids these problems but with a slight loss of in situ hybridization sensitivity.