Simultaneous triple fluorescence detection of mRNA localization, nuclear DNA, and apoptosis in cultured cells using confocal scanning laser microscopy.
Simultaneous triple fluorescence detection of mRNA localization, nuclear DNA, and apoptosis in cultured cells using confocal scanning laser microscopy.
复制标题
使用共焦扫描激光显微镜同时检测培养细胞中的 mRNA 定位、核 DNA 和细胞凋亡。
DOI：
10.1007/s004180050170
复制
发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Taatjes,DJ
中科院分区：
文献类型：
--
作者：
Davis,WP;Janssen,YM;Mossman,BT;Taatjes,DJ
我们描述了一种多荧光标记技术，用于同时检测培养细胞中的mRNA，核DNA和凋亡。使用地高辛标记的cRNA探针研究暴露于石棉或过氧化氢（H202）后发生凋亡的大鼠胸膜间皮细胞中原癌基因的表达。用抗地高辛单克隆一抗和荧光基团偶联抗小鼠二抗免疫定位检测杂交的cRNA探针。采用tdt介导的生物素- dutp镍端标记法（TUNEL）和链霉亲和素偶联的远红色荧光团同时对凋亡细胞进行鉴定，并用恶唑黄二聚体（YOYO-1）对细胞核DNA进行染色。通过共聚焦扫描激光显微镜，我们发现在暴露于石棉或H202后，tunel阳性细胞和非凋亡细胞的细胞质中c- junmrna表达增加。因此，该技术代表了一种有用的共聚焦扫描激光显微镜评估细胞凋亡相关基因表达的体内方法。
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 (H202). 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 increasedc-junmRNA expression within the cytoplasm of both TUNEL-positive and non-apoptotic cells following exposure to either crocidolite asbestos or H202. Thus, this technique represents a useful in vivo approach for evaluating apoptosis-associated gene expression with confocal scanning laser microscopy.