Fluorescence in situ hybridization (FISH) assay

The subcellular localization of UCA1 in cells was identified by FISH. According to the instructions of Ribo™ lncRNA FISH Probe Mix (Red) (RiboBio Co., Ltd, Guangdong, China), the specific methods were as follows: the slide was put into the 24-well culture plate, and the cells were seeded at 6 × 10⁴ cells/well and grown to 80% confluence. The slide was taken out, the cells were fixed with 1 mL 4% paraformaldehyde after cleaning with PBS. After being treated with protease K, glycine and phthalylation reagent, the cells were added with 250 μL pre-hybrid solution and incubated at 42 ℃ for 1 h. The pre-hybrid solution was absorbed, cells were added with 250 μL UCA1 (300 ng/mL) hybrid solution containing probe and hybridized overnight at 42 ℃. The nucleus was stained with phosphate-buffered saline with Tween (PBST)-diluted DAPI (ab104139, 1:100, Abcam, Shanghai, China), added to the 24-well culture plate, and stained for 5 min. The cells were sealed with anti-fluorescence quenching agent, observed and photographed under a fluorescence microscope (Olympus Optical Co., Ltd, Tokyo, Japan).