title: Combined analysis of in situ hybridization, cell cycle and structural markers using reflectance and immunofluorescence confocal microscopy.
abstract: A method for the simultaneous detection of mRNA by reflectance in situ hybridization (RISH), cell cycle and structural markers by immunofluorescence using confocal laser scanning microscopy is presented. The mRNA expression of two ras-related genes rhoB and rhoC was analysed in human breast cancer cell lines and human histological specimens (breast cancer tissues and skin biopsies). In breast cancer cell lines, the conditions were optimized to detect RNA-RNA hybrids and DNA synthesis after pulse-labelling with bromodeoxyuridine. Endonuclease-exonuclease digestion, which allows the accessibility to specific antibodies of halogenated pyrimidine molecules, was carried out following ISH. Finally, cytokeratin or vimentin staining was performed. The detection of signals, arising from 1-nm colloidal gold particles without silver enhancement, by reflectance confocal laser scanning microscopy is described. Bromodeoxybiridine DNA markers and cytokeratin/vimentin staining were detected concomitantly using different fluorochromes. To allow comparative expression of two related genes, the mRNA of rhoB and rhoC were detected using digoxigenin- or biotin-labelled riboprobes and, after 3-D imaging, a detailed analysis by optical horizontal (x, y) and vertical (x, z) sectioning was undertaken. The subsequent bromodeoxyuridine detection procedure permitted to us explore the specific transcription of these two genes during S and non-S phases. This method allows the identification and localization of several subcellular components in cells within a complex tissue structure and makes it possible to analyse further transcript localization in relation to the function of the encoded protein and to the cell cycle.
authors.affiliation: Laboratoire de Pharmacologie Expérimentale, Hôpital Saint-Louis, Paris, France.
authors.forename: G
authors.initials: G
authors.lastname: Linares-Cruz
authors.forename: G
authors.initials: G
authors.lastname: Millot
authors.forename: P
authors.initials: P
authors.lastname: De Cremoux
authors.forename: J
authors.initials: J
authors.lastname: Vassy
authors.forename: B
authors.initials: B
authors.lastname: Olofsson
authors.forename: J P
authors.initials: JP
authors.lastname: Rigaut
authors.forename: F
authors.initials: F
authors.lastname: Calvo
mesh_terms: D015415:Biomarkers; D001973:Bromodeoxyuridine; D002453:Cell Cycle; D019204:GTP-Binding Proteins / Q000235:genetics; D006801:Humans; D007091:Image Processing, Computer-Assisted; D017403:In Situ Hybridization / Q000379:methods*; D008565:Membrane Proteins / Q000235:genetics; D018613:Microscopy, Confocal / Q000295:instrumentation; D008856:Microscopy, Fluorescence / Q000379:methods; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D014407:Tumor Cells, Cultured; D020741:rho GTP-Binding Proteins; D020743:rhoB GTP-Binding Protein; D000075024:rhoC GTP-Binding Protein
chemical_list: D015415:Biomarkers; D008565:Membrane Proteins; D012333:RNA, Messenger; D019204:GTP-Binding Proteins; C066690:RHOC protein, human; D020741:rho GTP-Binding Proteins; D020743:rhoB GTP-Binding Protein; D000075024:rhoC GTP-Binding Protein; D001973:Bromodeoxyuridine