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.