2.5. RNA Fluorescence In Situ Hybridization (RNA-FISH) and Immunofluorescence

Cells on coverslips were fixed with 4% paraformaldehyde for 10 min at room temperature, then permeabilized with cold 2% acetone for 5 min, and overnight incubation in 70% ethanol. Cells were prehybridized in 30% formamide, 2X SSC buffer for 10 min at room temperature and then incubated for 2 h in a humidified chamber at 37 °C with hybridization buffer [2X SSC, 40% formamide, 0.02% BSA, 2mM vanadyl ribonucleoside (Sigma-Aldrich, St. Louis, MO, USA), 66 µg/mL yeast tRNA (Sigma-Aldrich, St. Louis, MO, USA) and 1 ng/µl Cy3-conjugated CAG₍₆₎ probe]. Preparations were washed in prehybridization buffer at 45 °C for 30 min, twice in 1X SSC at room temperature, and once in PBS. Cells were mounted on microscope slides with Vectashield antifade medium containing diamino-2-phenylindole (DAPI) (Vector Labs., Burlingame, CA, USA). For combine immunofluorescence and FISH, cells were incubated in 3% BSA for 15 min at room temperature after the post-hybridization wash step of the RNA FISH procedure, then incubated overnight at 4 °C with the primary anti-MBNL1 or MBNL2 antibodies (Abcam, Cambridge, UK. ab45899 and ab171551, respectively). After washing three times with PBS, preparations were incubated 1h at room temperature with fluorescein-conjugated anti-rabbit antibody (Vector Labs., Burlingame, CA, USA), counterstained with DAPI and mounted with Vectashield.