LINC00261 Subcellular Localization by FISH

The subcellular localization of LINC00261 was determined by FISH analysis as explained previously [56 (link)], following the instruction of Ribo^TM lncRNA FISH Probe Mix (Red) Kit (C10920, RiboBio, Shenzhen, China). Briefly, the cell slide was seeded in a 24-well plate at a density of 6 × 10⁴ cells/well until the cell confluence reached 60–70%. Subsequently, cells were fixed by 1 mL 4% paraformaldehyde at room temperature for 10 min and permeabilized with 1 mL precooled phosphate buffer saline (PBS) containing 0.5% Triton X-100 at 4 °C for 5 min. After PBS washing, cells in each well were sealed with 200 µL prehybridization solution at 37 °C for 30 min, and then hybridized with nucleotide probe against LINC00261 (GeneCreate, Wuhan, China) at 37 °C overnight without light. Cells were rinsed in Lotion I (4 × SSC, 0.1% Tween-20), Lotion II (2 × SCC), Lotion III (1 × SCC), respectively, and then washed with 1 × PBS three times (5 min for each). Finally, cells were stained in 4′,6-diamidino-2-phenylindole solution (1: 800) for 10 min, washed, and sealed with nail enamel. Five visual fields selected at random, cells were observed and imagined under a fluorescence microscope (Olympus, Japan).