SEMITHIN CRYOSECTIONS AS A TOOL TO PERFORM HIGH-RESOLUTION IMMUNOFLUORESCENCE AND IN-SITU HYBRIDIZATION ANALYSIS OF THE NERVOUS-TISSUE - A STUDY IN THE SUPRAOPTIC NUCLEUS

Immunofluorescence and fluorescence in in situ hybridization represent powerful approaches to correlate biochemical and molecular data with the structural organization of cells and tissues. However, the analysi s of tissues by fluorescence microscopy is limited by the fact that mo st methods currently used to preserve the morphological integrity of s ectioned samples at high resolution do not allow access of the labeled probes to the target molecules. Here we have made use of semithin cry osections obtained from rat supraoptic nucleus to perform immunofluore scence with antibodies directed against cytoplasmic and nuclear antige ns, as well as fluorescence in situ hybridization with antisense oligo nucleotide probes complementary to the poly(A) tail of mRNA and to spe cific mRNAs. In addition, DNA was visualized by incubation of sections with digoxigenin-labeled nucleotides in the presence of Escherichia c oil DNA polymerase I. The high resolution of this DNA staining in comb ination with immunolabeling for nuclear antigens provides a powerful t ool to analyze the structural and functional compartmentalization of n euronal cell nuclei. The major conclusion from this study is that perf orming fluorescence microscopy on 1 mu m-thick cryosections provides a n important tool to accurately localize proteins, DNA and RNA within n ervous tissue in general and particularly in the model of supraoptic n ucleus. Moreover, the cryosectioning technique appears particularly su ited to the study of the localization of specific mRNA. species in the neuronal cytoplasm and represents a useful approach to addressing the functional significance of mRNA localization in protein targeting. (C ) 1997 Elsevier Science B.V.