No 1.5 thickness 12 mm round glass coverslips

STED imaging was performed using a Leica TCS SP8 STED 3X microscope. Isolated mouse ventricular myocytes were fixed with 4% paraformaldehyde on No. 1.5-thickness/12-mm-round glass coverslips (Thomas Scientific, Swedesboro, NJ, USA) for 20 minutes at room temperature (RT) and washed three times with phosphate-buffered saline (PBS) containing 0.05% Triton-X. Non-specific antibody binding was blocked with 10% bovine serum albumin (BSA) in 0.1% PBS with Tween 20 PBST for 1 hour at RT. For double-labeling experiments, cells were first treated with a primary antibody (anti-GFP antibody and anti-α-actinin antibody, incubated overnight at 4°C, followed by application of secondary antibodies sequentially with Oregon Green 488 goat-anti-rabbit secondary antibody (Thermo Fisher Scientific) followed by Rhodamine goat-anti-mouse secondary antibody (Thermo Fisher Scientific) for 1-hour incubation at RT For STED imaging, ProLong Gold Antifade Mountant (Thermo Fisher Scientific) was used to minimize photobleaching. Negative controls with only antigenic peptides and secondary antibodies were performed for all experiments.