Anti mlc 2v

EBs or ESC-CMs obtained on day 5+3 of differentiation were digested into single cells with Collagenase II (Invitrogen, USA). Cells were fixed with 4% paraformaldehyde for 1 h and then treated with 10% FBS for another 1 h to block non-specific antigens. Cells were then incubated in PBS containing one of the primary antibodies overnight at 4 ºC. The primary antibodies included mouse monoclonal anti-α-Actinin (Sigma Aldrich, A-7811, USA), rabbit polyclonal anti-MLC-2v (Abcam, ab-79935, USA), mouse monoclonal anti-Connexin 43 (Abcam, ab-79010, USA). After washing with PBS twice, cells were incubated with Alexa fluor 488-conjugated anti-mouse IgG or phycoerythrin-conjugated anti-rabbit IgG (Invitrogen, USA) for 1 h, and then suspended in 0.5 ml 1% BSA and analyzed on a FACScan flow cytometry (Becton-Dickson, Sparks, USA). The fluorochrome was detected at 530 nm in the FL-1 and 575nm in the FL-2 channel. Each plot represented 10,000 viable cells (nonviable cells were excluded from FACS analysis by appropriate gating). Untreated cells and cells lacking primary antibody were used as negative controls. In addition, isotype controls were used to assess the level of non-specific antibody binding 24 (link). All data analyses were carried out by using Cell Quest software.