COMBINED LIGHT AND FLUORESCENT MICROSCOPIC IMAGING OF NUCLEOLAR ORGANIZER REGIONS AND CELLULAR RIBOSOMAL-RNA AS DETECTED BY FLUORESCENT IN-SITU HYBRIDIZATION

A method for combined Light and fluorescent microscopic imaging of nuc leolar organizer regions and cellular rRNA is described. Nucleolar org anizer regions were detected by silver staining (Ag-NOR), and rRNA was detected by fluorescent in situ hybridization (FISH). MG-63 human fib rosarcoma cells were silver stained prior to in situ hybridization. To quantitate Ag-NOR within individual cells, brightfield images were di gitized, and the total Ag-NOR area/nucleus was determined. Fluorescent images were digitized, and the total cellular fluorescence was calcul ated after correction for nonuniformity of illumination. By using this method, it was shown that the Ag-NOR procedure did not signifi-cantly affect the fluorescence intensity related to FISH. Furthermore, the h ybridization procedure did not interfere with quantitation of Ag-NOR. With this method, both Ag-NOR and rRNA product can be quantitated with in the same cell. Because the relationship of rRNA content to cell pro liferation is well established, correlation to quantitative Ag-NOR par ameters within individual cells will contribute to the better definiti on of the relationship of quantitative Ag-NOR indices with cellular pr oliferation. (C) 1995 Wiley-Liss, Inc.