Reflectance in situ hybridization (RISH): detection, by confocal reflectance laser microscopy, of gold-labelled riboprobes in breast cancer cell lines and histological specimens

• Published in: Journal of microscopy (Oxford) Vol. 173; no. Pt 1; p. 27
• Main Authors: Linares-Cruz, G, Rigaut, J P, Vassy, J, De Oliveira, T C, De Cremoux, P, Olofsson, B, Calvo, F
• Format: Journal Article
• Language: English
• Published: England 01.01.1994
• Subjects: Antibodies, Monoclonal; Antisense Elements (Genetics); Blotting, Northern; Breast Neoplasms - genetics; Breast Neoplasms - pathology; DNA, Neoplasm - analysis; DNA, Neoplasm - genetics; Female; Gene Expression Regulation, Neoplastic - genetics; GTP-Binding Proteins - genetics; Humans; Immunohistochemistry; In Situ Hybridization - methods; Keratins - analysis; Keratins - genetics; Lasers; Membrane Proteins - genetics; Microscopy - methods; rho GTP-Binding Proteins; rhoB GTP-Binding Protein; rhoC GTP-Binding Protein; RNA Probes; Tumor Cells, Cultured; Vimentin - analysis; Vimentin - genetics
• ISSN: 0022-2720; 1365-2818
• DOI: 10.1111/j.1365-2818.1994.tb03425.x

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.