Dual FISH and ICC Assay
Following PFA fixation, cells were washed with PBS and then incubated in blocking solution containing 10% donkey serum, 0.5% Triton X-100, 3% BSA, and 0.4 U/μL RNase inhibitor in PBS for 1 hr at room temperature. Cells were subsequently incubated with mouse anti-MBNL1 antibody (1:1,000 in blocking solution; kindly provided by Thornton Lab, Chicago) overnight at 4°C. Following primary antibody incubation, cells were washed with PBS and incubated with secondary anti-mouse antibody conjugated with Alexa Fluor 488 (1:1,000 in 0.5% Triton X-100, 3% BSA) for 1 hr at room temperature. Cells were again washed with PBS and post-fixed in 4% PFA in PBS for 10 min. FISH was subsequently performed, in accordance with methods stated above.