RRC ID 33960
Author Davis WP, Janssen YM, Mossman BT, Taatjes DJ.
Title Simultaneous triple fluorescence detection of mRNA localization, nuclear DNA, and apoptosis in cultured cells using confocal scanning laser microscopy.
Journal Histochem Cell Biol
Abstract 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.
Volume 108(4-5)
Pages 307-11
Published 1997-10
DOI 10.1007/s004180050170
PMID 9387922
MeSH Animals Apoptosis* Asbestos, Crocidolite Benzoxazoles / chemistry Carbocyanines / chemistry Cell Nucleus / chemistry Cells, Cultured DNA / analysis* Fluorescence Fluorescent Dyes / chemistry In Situ Hybridization, Fluorescence / methods* Microscopy, Confocal / methods* Proto-Oncogene Proteins c-fos / genetics Proto-Oncogene Proteins c-jun / genetics Quinolinium Compounds / chemistry RNA, Messenger / analysis* Rats Ribonucleases / metabolism Water
IF 2.64
Times Cited 15
DNA material pBS rcjun-1 (RDB01130)