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Reflectance in situ hybridization (RISH): detection,by confocal reflectance laser microscopy,of gold-labelled riboprobes in breast cancer cell lines and histological specimens
Authors:G LINARES-CRUZ  J P RIGAUT  J VASSY  T C De OLIVEIRA  P De CREMOUX  B OLOFSSON  F CALVO
Abstract:A method for reflectance in situ hybridization (RISH) is presented. The importance of the method is demonstrated by results obtained on cytological and histological breast cancer specimens. Scattering reflectance signals from 1-nm colloidal-gold particles after RNA/RNA in situ hybridization, using digoxigenin-labelled riboprobes, were detected by confocal scanning laser microscopy. The mRNA expression of two ras-related 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 individual 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 method allows the identification of several cellular constituents in an heterogeneous tissue structure, as demonstrated by the simultaneous detection of rho B (or rho C) by reflectance and of DNA, cytokeratin and/or vimentin by fluorescence.
Keywords:Confocal scanning laser microscopy  in situ hybridization  scattering  reflectance  gold immunolabelling  multi-imaging  breast tumours  rho genes
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