COMBINED ANALYSIS OF IN-SITU HYBRIDIZATION, CELL-CYCLE AND STRUCTURALMARKERS USING REFLECTANCE AND IMMUNOFLUORESCENCE CONFOCAL MICROSCOPY

A method for the simultaneous detection of mRNA by reflectance in situ hybridization (RISH), cell cycle and structural markers by immunofluo rescence using confocal laser scanning microscopy is presented. The mR NA expression of two ras-related genes rhoB and rhoC was analysed in h uman breast cancer cell lines and human histological specimens (breast cancer tissues and skin biopsies). In breast cancer cell lines, the c onditions were optimized to detect RNA-RNA hybrids and DNA synthesis a fter pulse-labelling with bromodeoxyuridine. Endonuclease-exonuclease digestion, which allows the accessibility to specific antibodies of ha logenated pyrimidine molecules, was carried out following ISH. Finally , cytokeratin or vimentin staining was performed. The detection of sig nals, arising from 1-nm colloidal gold particles without silver enhanc ement, by reflectance confocal laser scanning microscopy is described. Bromodeoxybiridine DNA markers and cytokeratin/vimentin staining were detected concomitantly using different fluorochromes. To allow compar ative 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 verti cal (x, z) sectioning was undertaken. The subsequent bromodeoxyuridine detection procedure permitted to us explore the specific transcriptio n 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 analy se further transcript localization in relation to the function of the encoded protein and to the cell cycle.