Simultaneous triple fluorescence detection of mRNA localization, nuclear DNA, and apoptosis in cultured cells using confocal scanning laser microscopy.
We describe a multifluorescence labeling technique for simultaneous detection of mRNA, nuclear DNA, and apoptosis in cultured cells. Digoxigenin-labeled cRNA probes were used to study proto-oncogene expression in rat pleural mesothelial cells undergoing apoptosis following exposure to crocidolite asbestos or hydrogen peroxide (H2O2). Hybridized cRNA probe was detected by immunolocalization with an anti-digoxigenin monoclonal primary and fluorophore-conjugated anti-mouse secondary antibody. Cells undergoing apoptosis were simultaneously identified by the TdT-mediated biotin-dUTP nick-end labeling (TUNEL) method and a streptavidin-conjugated far-red fluorophore, and nuclear DNA was stained with oxazole yellow dimer (YOYO-1). With confocal scanning laser microscopy, we demonstrated increased c-jun mRNA expression within the cytoplasm of both TUNEL-positive and non-apoptotic cells following exposure to either crocidolite asbestos or H2O2. Thus, this technique represents a useful in vivo approach for evaluating apoptosis-associated gene expression with confocal scanning laser microscopy.
Davis
W P
WP
Department of Pathology, University of Vermont, Burlington 05405, USA.
Janssen
Y M
YM
Mossman
B T
BT
Taatjes
D J
DJ
D000818:Animals; D017209:Apoptosis; D017638:Asbestos, Crocidolite; D001583:Benzoxazoles / Q000737:chemistry; D002232:Carbocyanines / Q000737:chemistry; D002467:Cell Nucleus / Q000737:chemistry; D002478:Cells, Cultured; D004247:DNA / Q000032:analysis*; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D018613:Microscopy, Confocal / Q000379:methods*; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics; D016755:Proto-Oncogene Proteins c-jun / Q000235:genetics; D011806:Quinolinium Compounds / Q000737:chemistry; D012333:RNA, Messenger / Q000032:analysis*; D051381:Rats; D012260:Ribonucleases / Q000378:metabolism; D014867:Water
D001583:Benzoxazoles; D002232:Carbocyanines; D005456:Fluorescent Dyes; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D011806:Quinolinium Compounds; D012333:RNA, Messenger; C089187:cyanine dye 3; D014867:Water; D017638:Asbestos, Crocidolite; C075296:1,1'-((4,4,7,7-tetramethyl)-4,7-diazaundecamethylene)bis-4-(3-methyl-2,3-dihydro(benzo-1,3-oxazole)-2-methylidene)quinolinium; D004247:DNA; D012260:Ribonucleases