Immunofluorescence
Adjacent sections from each group were double-labeled with connexin 43 (Cx43) and HCN4 antibodies, connexin 40 (Cx40) and connexin 45 (Cx45) antibodies, and Nav1.5 and Cav3.1 antibodies. Briefly, sections were fixed in 4% paraformaldehyde solution for 10-15 minutes, washed three times with PBS (pH 7.4), treated with 0.3% Triton X-100 and blocked with 5% normal donkey serum for one hour at room temperature. Sections were incubated with the following primary antibodies in blocking solution at 4°C overnight: mouse anti-Cx45 (1:100; Abcam Inc.), goat anti-Cx40 (1:100; Santa Cruz Biotechnology, Inc.), rabbit anti-Cx43 (1:100; Cell Signaling Technology, Inc.), rabbit anti-Nav1.5 (1:200; Alomone Labs, Inc.), rabbit anti-Cav3.1 (1:200; Alomone Labs, Inc.) and goat anti-HCN4 (1:50; Santa Cruz Biotechnology, Inc.). Subsequently, sections were extensively washed in PBS and incubated in a diluted solution for one hour at room temperature with the following secondary antibodies: DyLight 594-conjugated donkey anti-mouse (1:100; EarthOx, San Francisco, CA, USA), DyLight 488-conjugated donkey anti-goat (1:100; EarthOx, San Francisco, CA, USA), DyLight 488-conjugated donkey anti-rabbit (1:100; EarthOx, San Francisco, CA, USA), DyLight 594-conjugated donkey anti-goat (1:100; EarthOx, San Francisco, CA, USA) and DyLight 594-conjugated donkey anti-rabbit (1:100; EarthOx, San Francisco, CA, USA). Nuclei were stained with Draq5 (1:10000; Cell Signaling Technology) within the secondary antibody incubation step. Sections were mounted in mounting medium and imaged using a fluorescence microscope (LEICA DM4000B). Signal intensity measurements were then obtained using Image-Pro Plus 6.0 software after background correction.