Prolong gold antifade

HEK293 cells were seeded on glass coverslips in tissue culture dishes 35×10 mm (Sarstedt, 83.1800) and grown to 90% confluence for 48 h in DMEM supplemented with GlutaMax (Life Technologies), 10% fetal calf serum and 1% penicillin/streptomycin (PAA). Confluent cells were transfected with Turbofect (Thermo Scientific) according to the manufacturer’s instructions. After 24 h of protein expression, cells were washed with PBS buffer and fixed with 4% PFA for 15 min at room temperature. Afterwards, cells were washed with PBS buffer and mounted on microscopy slides with ProLong Gold Antifade (Sigma, 36930). Localization of DdTMEM16-GFP protein in HEK293 cells was analyzed by imaging fluorescence with a Leica SPE confocal microscope (Leica, TCS SPE).

Immunocytochemical staining of the cultured cardiomyocytes was performed using the following antibodies. Using a typical process, the cardiomyocytes were placed in a 4% formaldehyde-dissolved Dulbecco’s phosphate-buffered saline (DPBS) solution for 20 min at room temperature and, then, washed three times with DPBS. Then, the cardiomyocytes were permeabilized with 0.1% Triton X-100 (Sigma–Aldrich) in DPBS for 5 min and blocked for 30 min in 3% bovine serum albumin (BSA) (Sigma–Aldrich). Next, the cardiomyocytes were incubated with the primary antibodies, including mouse monoclonal α-sarcomere actinin (Abcam), and Troponin-T (TnT) (Abcam), which were diluted to a ratio of 1:100 with 1% BSA solution, for 90 min at room temperature. The secondary antibodies (Alexa Fluor 488 Goat anti-mouse lgG conjugate and Alexa Fluor 568 Goat anti-rabbit lgG+ (H+L) conjugate) were diluted to 1:200 in the same blocking solution that was used for the primary antibodies and the cardiomyocytes were incubated in them for 90 min at room temperature. Finally, the samples were mounted using coverslips and ProLong Gold Antifade (Sigma–Aldrich). Finally, the cardiomyocytes were analyzed by inverted confocal laser scanning microscopy (Leica TCS SP5 XCLSM, Germany).