SIMULTANEOUS TRIPLE FLUORESCENCE DETECTION OF MESSENGER-RNA LOCALIZATION, NUCLEAR-DNA, AND APOPTOSIS IN CULTURED-CELLS USING CONFOCAL SCANNING LASER MICROSCOPY

We describe a multifluorescence labeling technique for simultaneous de tection of mRNA, nuclear DNA, and apoptosis in cultured cells. Digoxig enin-labeled cRNA probes were used to study proto-oncogene expression in rat pleural mesothelial cells undergoing apoptosis following exposu re to crocidolite asbestos or hydrogen peroxide (H2O2). Hybridized cRN A probe was detected by immunolocalization with an anti-digoxigenin mo noclonal primary and fluorophore-conjugated anti-mouse secondary antib ody. Cells undergoing apoptosis were simultaneously identified by the TdT-mediated biotin-dUTP nick-end labeling (TUNEL) method and a strept avidin-conjugated far-red fluorophore, and nuclear DNA was stained wit h oxazole yellow dimer (YOYO-1). With confocal scanning laser microsco py, we demonstrated increased c-jun mRNA expression within the cytopla sm of both TUNEL-positive and non-apoptotic cells following exposure t o either crocidolite asbestos or H2O2. Thus, this technique represents a useful in vivo approach for evaluating apoptosis-associated gene ex pression with confocal scanning laser microscopy.