Ab2912

Intact cardiobundles or 10 μm thick cardiobundles cross-sections were fixed in 2% paraformaldehyde (PFA) and immunostained as previously described [9 (link), 30 (link)]. Briefly, intact or sectioned tissue samples were permeabilized with 0.5% Triton-X for 30 min, then blocked with a blocking solution (1% bovine serum albumin and 10% chicken serum in PBS) at 4 °C overnight. Primary antibodies were diluted in blocking solution and incubated with samples overnight at 4 °C, followed by secondary antibody incubation at room temperature for 2 hours. The primary antibodies used included Sarcomeric α-actinin (SAA, Sigma A7811, 1:200), Connexin-43 (Abcam Ab11370, 1:200), N-cadherin (Abcam Ab12221, 1:400), Vimentin (Abcam Ab92547, 1:500), Caveolin 3 (Abcam Ab2912, 1:200), Ki67 (Santa Cruz sc-7844, 1:300), Nkx2.5 (Santa Cruz sc-8697 1:100), and RyR2 (Thermo MA3916, 1:150). Samples were imaged using Zeiss 510 inverted confocal microscope and image analysis was performed using image J software.
To stain cell membranes for visualization of T-tubules, live cardiobundles were washed in Tyrode’s solution, then incubated for 5 min in 10 μM di-4 ANEPPS dye at room temperature, and subsequently rinsed and incubated for additional 5 min in Tyrode’s solution. Di-4 stained cardiobundles were imaged on a Zeiss 510 inverted confocal using a 40× NA/1.3 oil objective.