Rabbit polyclonal anti connexin 43

NRCMs seeded on bioprinted constructs and cultured until maximum beating rate were obtained (day 7). This ensured that sarcomeres in the beating NRCMs were mature and fully developed. After gently washing with DPBS (pH 7.4), the samples were fixed using 4% v/v paraformaldehyde for 20 min at RT. The fixed cells were permeabilized with DPBS (pH 7.4) consisting of 0.1% v/v Triton X-100 for 20 min, washed thoroughly and then blocked using a blocking buffer (1% BSA, 0.2% Tween in DPBS) for 1 h. The samples were incubated at 4 °C overnight with primary antibodies such as rabbit polyclonal anti-connexin 43 and mouse monoclonal anti-sarcomeric α−actinin (1:100, Abcam). Subsequently, the samples were washed thrice with DPBS in 10 min intervals. For determining the maturation of vascularized myocardial tissue, the constructs were permeabilized and kept for blocking as described above. Furthermore, they were incubated at 4 °C overnight with primary antibodies such as rabbit polyclonal anti-troponin T (1:100, Abcam) and rabbit polyclonal CD31 (1:100, Abcam). Furthermore, the samples were treated by the corresponding secondary antibodies for 3 h at 1:200 dilutions. Nuclei were stained by incubating with 4’,6-diamidino-2-phenylindole (DAPI; 1:500) for 1.5 h. The stained samples were then visualized using a confocal microscope.