REFLECTANCE IN-SITU HYBRIDIZATION (RISH) - DETECTION, BY CONFOCAL REFLECTANCE LASER MICROSCOPY, OF GOLD-LABELED RIBOPROBES IN BREAST-CANCERCELL-LINES AND HISTOLOGICAL SPECIMENS

A method for reflectance in situ hybridization (RISH) is presented. Th e importance of the method is demonstrated by results obtained on cyto logical and histological breast cancer specimens. Scattering reflectan ce signals from 1-nm colloidal-gold particles after RNA/RNA in situ hy bridization, using digoxigenin-labelled riboprobes, were detected by c onfocal scanning laser microscopy. The mRNA expression of two ras-rela ted genes, rho B and rho C, was analysed in human histological breast cancer specimens and in human breast cancer cell lines. Horizontal (x, y) and vertical (z) optical sections after three-dimensional imaging were used for visualization. A marked heterogeneity (between individua l cells and between specimens) was noted for the expression of the rho B gene, both in cytological and in histological samples. On the other hand, rho C was always expressed and showed no heterogeneity. This me thod allows the identification of several cellular constituents in an heterogeneous tissue structure, as demonstrated by the simultaneous de tection of rho B (or rho C) by reflectance and of DNA, cytokeratin and /or vimentin by fluorescence.