Anti alpha actinin

Immunostaining was performed using anti-OCT4, anti-SEEA4, anti-TRA-1-60, anti-SOX2 (Thermo Fisher Scientific, Waltham, MA, USA), anti-NKX2.5, anti-TNNT2/cTNT, anti-alpha-actinin (Sigma-Aldrich, St. Louis, MO, USA, A7811), and MYL2 (ProteinTech, Rosemont, IL, USA, 10906-1-AP) as primary antibodies. The procedure was performed on a 24-well culture dish with 60–70% cell confluence. The cells were washed with PBS twice, followed by fixing with 4% formaldehyde in Dulbecco’s PBS (DPBS) for 15 min. After additional washing with PBS, the cells were permeabilized by 1% saponin in DPBS for 5 min and blocked with 3% BSA in DPBS for 30 min at room temperature. The cells were incubated with the designed primary antibody overnight at 4 °C. Then, the cells were washed thrice with PBS and incubated with the designed secondary antibodies for 1 h. The chamber was washed thrice with PBS, and nuclear counterstaining was carried out with NucBlue™ Fixed Cell ReadyProbes™ Reagent (4′,6-diamidino-2-phenylindole (DAPI), Thermo Fisher Scientific, Waltham, MA, USA) for 1 min. After washing with PBS twice, images were captured with a Zeiss Axioplan 2 Imaging microscope with Plan-NEOPLUAR 10×/0.75 objective lens.

For cardiomyocyte characterization, immunostaining was performed using anti-cTNT (Abcam; 1:100) and anti-alpha-actinin (Sigma-Aldrich; 1:200) as primary antibodies and Alexafluor-488-cojugated anti-mouse IgG as a secondary antibody. Briefly, cells were washed with PBS (Life technologies), fixed with 4 % PFA (Sigma-Aldrich) for 15 min and permeabilized in PBS containing 0.1 % Triton X-100 (Sigma-Aldrich) and 5 % FCS (Life techologies) for 30 min. Cells were then incubated overnight with the primary antibodies. Next day, secondary antibody Alexafluor-488-cojugated anti-mouse IgG (1:1,000; Life technologies) were used to detect and visualize the primary antibodies. All antibodies were diluted in blocking solution. Similar protocol was also used for the staining using ISL1 (Biorbyt; 1:200) antibody. Micrographs were taken with an Axiovert 200 M microscope (Carl Zeiss).