The AGAP2-ASA and miR-9-5p localization in cells was determined using the FISH technique. RiboTM lncRNA FISH Probe Mix (Red) (Ruibo Biotechnology, China) was used according to the manufacturer's instructions [47 (link)]. The specific method was as follows: coverslips were placed in a 6-well culture plate, and the test cells (1 × 105 cells/well) were seeded into the wells and incubated for 1 day to reach a cell fusion rate of around 80%. The coverslips were removed, washed with PBS, and fixed with 1 mL of 4% paraformaldehyde at room temperature. After treatment with proteinase K (2 μg/mL), glycine, and acetylation reagent, 250 μL of pre-hybridization solution was added and incubated at 42 °C for 1 h. The pre-hybridization solution was removed, and 250 μL of hybridization solution containing the probes (300 ng/mL) was added and hybridized overnight at 42 °C. After washing three times with PBST, the nuclei were stained with DAPI diluted in PBST (1:800) and incubated for 5 min in a 24-well culture plate. The samples were then washed three times with PBST for 3 min each. Finally, the coverslips were mounted with an anti-fluorescence quenching agent and observed and photographed using a laser confocal microscope (Leica, TCS-SP8 SR, Germany) with five fields of view selected for analysis.
Free full text: Click here
