Combined light and fluorescent microscopical imaging of nucleolar organizer regions and cellular rRNA as detected by fluorescent in situ hybridization.
A method for combined light and fluorescent microscopic imaging of nucleolar organizer regions and cellular rRNA is described. Nucleolar organizer regions were detected by silver staining (Ag-NOR), and rRNA was detected by fluorescent in situ hybridization (FISH). MG-63 human fibrosarcoma cells were silver stained prior to in situ hybridization. To quantitate Ag-NOR within individual cells, brightfield images were digitized, and the total Ag-NOR area/nucleus was determined. Fluorescent images were digitized, and the total cellular fluorescence was calculated after correction for nonuniformity of illumination. By using this method, it was shown that the Ag-NOR procedure did not significantly affect the fluorescence intensity related to FISH. Furthermore, the hybridization procedure did not interfere with quantitation of Ag-NOR. With this method, both Ag-NOR and rRNA product can be quantitated within the same cell. Because the relationship of rRNA content to cell proliferation is well established, correlation to quantitative Ag-NOR parameters within individual cells will contribute to the better definition of the relationship of quantitative Ag-NOR indices with cellular proliferation.
Pajor
L
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Department of Surgery, University of Massachusetts Medical Center, Worcester, USA.
Honeyman
T W
TW
D005354:Fibrosarcoma; D006801:Humans; D007091:Image Processing, Computer-Assisted; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008856:Microscopy, Fluorescence; D009697:Nucleolus Organizer Region / Q000648:ultrastructure*; D012335:RNA, Ribosomal / Q000648:ultrastructure*; D016622:Silver Staining / Q000379:methods*; D014407:Tumor Cells, Cultured
D012335:RNA, Ribosomal