Reflectance in situ hybridization (RISH): detection, by confocal reflectance laser microscopy, of gold-labelled riboprobes in breast cancer cell lines and histological specimens.
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.
Linares-Cruz
G
G
Laboratoire de Pharmacologie, Expérimentale, Hôpital Saint-Louis, Université Paris 7, France.
Rigaut
J P
JP
Vassy
J
J
De Oliveira
T C
TC
De Cremoux
P
P
Olofsson
B
B
Calvo
F
F
D000911:Antibodies, Monoclonal; D016375:Antisense Elements (Genetics); D015152:Blotting, Northern; D001943:Breast Neoplasms / Q000235:genetics* / Q000473:pathology; D004273:DNA, Neoplasm / Q000032:analysis / Q000235:genetics; D005260:Female; D019204:GTP-Binding Proteins / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics; D006801:Humans; D007150:Immunohistochemistry; D017403:In Situ Hybridization / Q000379:methods*; D007633:Keratins / Q000032:analysis / Q000235:genetics; D007834:Lasers; D008565:Membrane Proteins / Q000235:genetics; D008853:Microscopy / Q000379:methods*; D015347:RNA Probes; D014407:Tumor Cells, Cultured; D014746:Vimentin / Q000032:analysis / Q000235:genetics; D020741:rho GTP-Binding Proteins; D020743:rhoB GTP-Binding Protein; D000075024:rhoC GTP-Binding Protein
D000911:Antibodies, Monoclonal; D016375:Antisense Elements (Genetics); D004273:DNA, Neoplasm; D008565:Membrane Proteins; D015347:RNA Probes; D014746:Vimentin; D007633:Keratins; D019204:GTP-Binding Proteins; C066690:RHOC protein, human; D020741:rho GTP-Binding Proteins; D020743:rhoB GTP-Binding Protein; D000075024:rhoC GTP-Binding Protein