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 analysis of tissues by fluorescence microscopy is limited by the fact that most methods currently used to preserve the morphological integrity of sectioned samples at high resolution do not allow access of the labeled probes to the target molecules. Here we have made use of semithin cryosections obtained from rat supraoptic nucleus to perform immunofluorescence with antibodies directed against cytoplasmic and nuclear antigens, as well as fluorescence in situ hybridization with antisense oligonucleotide probes complementary to the poly(A) tail of mRNA and to specific mRNAs. In addition, DNA was visualized by incubation of sections with digoxigenin-labeled nucleotides in the presence of Escherichia coli DNA polymerase I. The high resolution of this DNA staining in combination with immunolabeling for nuclear antigens provides a powerful tool to analyze the structural and functional compartmentalization of neuronal cell nuclei. The major conclusion from this study is that performing fluorescence microscopy on 1 micron-thick cryosections provides an important tool to accurately localize proteins, DNA and RNA within nervous tissue in general and particularly in the model of supraoptic nucleus. Moreover, the cryosectioning technique appears particularly suited 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.
Lafarga
M
M
Department of Anatomy and Cell Biology, Faculty of Medicine, University of Cantabria, Santander, Spain. lafargam@medi.unican.es
Berciano
M T
MT
Carmo-Fonseca
M
M
D000818:Animals; D001710:Biotin; D016616:Cryoultramicrotomy / Q000379:methods*; D004247:DNA / Q000378:metabolism; D019085:Fluorescent Antibody Technique, Direct; D017403:In Situ Hybridization; D008297:Male; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009474:Neurons / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013495:Supraoptic Nucleus / Q000033:anatomy & histology* / Q000166:cytology / Q000378:metabolism; D016707:Tissue Fixation
D012313:RNA; D001710:Biotin; D004247:DNA