Fluorescence in situ hybridization (FISH)

The subcellular localization of SLCO4A1-AS1 in CD133⁺CD44⁺ colon cancer cells was detected using the FISH Kit ({"type":"entrez-nucleotide","attrs":{"text":"C10910","term_id":"1535981","term_text":"C10910"}}C10910, Guangzhou RiboBio Co., Ltd., Guangzhou, Guangdong, China). HCT116 cells in logarithmic growth phase were transferred onto slides in a 24-well plate (about 6 × 10⁴ cells/well). When cell confluence reached ~60–70%, the cells were fixed with 4% paraformaldehyde at room temperature for 10 min, and permeabilized with 1 mL pre-cooled Triton X-100 at 4 °C for 5 min. Cells were blocked with 200 μL pre-hybrid solution at 37 °C for 30 min. After that the cells were incubated with 2.5 μL hybridization solution containing 20 μM FISH Probe Mix overnight at 37 °C in dark, washed with solution I, solution II and solution III respectively at 42 °C. The cells were washed with 1× PBS for 5 min at room temperature. Next, cells were stained using 6-diamidino-2-phenylindole for 10 min and washed in 1× PBS for three times (5 min each time), after which the cells mounted and observed. The specific probe for lncRNA SLCO4A1-AS1 was synthesized by Guangzhou RiboBio Co., Ltd. (Guangzhou, Guangdong, China).