RNA fluorescent in situ hybridization

Human skin fibroblasts were fixed with 4 % PFA (Sigma Aldrich)at RT for 15 min. Slides were prehybridized in 30% volume of formamide and 2× SSC buffer for 10 min at 37°C and hybridized overnight in the solution containing 30% volume of formamide, 2× SSC buffer, 1 mg/ml tRNA, 0.02% BSA, 2 mM EDTA, and Cy3-labeled CAG probe. The incubation was followed by three washes with SSC buffer and DAPI staining for 15 min. After DAPI staining, the fluorescent signals were analyzed using the Laser Scanning Confocal Microscopy under the same brightness and exposure time.