Ab11370

Six hours after exposure, the round coverslips were placed in a 24-well plate, and 1 mL of 4% paraformaldehyde (P0099, Beyotime, Shanghai, China) was added to each well for 15 min; 1 mL of permeabilization buffer with Trixton-X-100 (P0096, Beyotime, Shanghai, China) was added to rupture the membrane for 10 min; 1 mL of 10% goat serum (diluted 1:10 in PBS; C0265, Beyotime, Shanghai, China) was used as blocking solution at room temperature for 1 h. Then, 200 μL of Cx43 primary antibody (1:100 dilution in 10% goat serum; ab11370, Abcam, Cambridge, UK) was added and incubated for 1 h at room temperature; then, fluorescently labeled secondary antibody (1:100 dilution in 10% goat serum; ab150080, Abcam, Cambridge, UK) was added and incubated in the dark at room temperature for 1 h. The round coverslips were removed and put on slides, and after the DAPI mounting medium (H-1200, Vector Laboratories, Newark, CA, USA) was added, the square coverslips were covered and fixed with nail polish. The images were observed under a fluorescence microscope (Nikon, Tokyo, Japan). The optical density values were semi-quantified, and statistical analysis was conducted. The cells were washed with DPBS (14190144, Gibco, Carlsbad, CA, USA) 3 times, 3 min each time, before each medium change.

The tissue sections (10 μm thick) were fixed with methanol/acetone 1:1 for 20 min at −20 °C, after which the tissue was used for immunofluorescence analysis. Staining was performed using the following primary antibodies: sarcomere α-actinin (ab9465, Abcam, UK) and Cx43 (ab11370, Abcam, UK). Confocal images were taken using a Leica TCS SP5 DMI6000 inverted microscope (with the TCS SP5 confocal scanner and acquisition software LAS-AF; Leica Microsystems, Mannheim, Germany). The Zeiss AxioScan Z1 digitized the immunofluorescence images.