Ab79935

Embryos were fixed overnight with 4% PFA at 4 °C; permeabilized for 10 h in 2% Triton X‐100 in PBS at room temperature; blocked overnight in 0.1% Tween‐20, 0.01% Triton X‐100, and 1% BSA in PBS at 4 °C; and incubated with primary antibodies at 4 °C overnight. The primary antibodies were PAX6 (1:100, AF8150, R&D Systems), CD31 (1:100, ab222783, Abcam), GATA4 (1:100, sc‐25310, Santa Cruz), myosin light chain (1:100, ab79935, Abcam), myosin heavy chain (1:100, 53‐6503‐82, eBioscience). The secondary antibodies were incubated overnight at 4 °C. The secondary antibodies were Alexa 488 goat anti‐rabbit (1:500, A11034, Invitrogen) and Alexa Fluor 647 donkey anti‐sheep (1:500, A21448, Invitrogen). Nuclear staining and incubation in were performed 10 µg mL⁻¹ Hoechst 33 342 (H3570, Invitrogen) for 10 min at room temperature. The embryos were imaged on confocal microscopes Leica SP8, and Zeiss LSM780 and Imaris 9.0.2 software was used to construct the 3D images/videos.

For intracellular staining of cardiac troponin T (cTnT) and myosin light chain 2 ventricle (MLC-2V), dissociated cardiomyocytes were fixed with 4% paraformaldehyde, permeabilized in PBS with 5% FCS and 0.5% Saponin, and washed again with PBS containing 0.5% BSA (Sigma). Cells were then stained overnight (4 °C) with the unconjugated primary antibodies targeting cTnT (MA5-12960, Thermo-Fisher Scientific) and MLC-2V (ab79935, Abcam) in FACS buffer consisting of PBS with 5% fetal bovine serum (FBS, Gibco). Cells were then washed again and stained with secondary antibodies (diluted in PBS with 5% FCS) for 1 h at 4 °C. Finally, cells were evaluated with the LSR FortessaII flow cytometer (BD-Biosciences), and FlowJo software.

Isolated iPSC-CMs were dissociated on to glass coverslips coated with 0.1% gelatin and then cultured for a further 2–12 days as described above. Cells were washed with ice-cold PBS supplemented with 1 mM MgCl₂ and 0.1 mM CaCl₂ (PBS⁺) then incubated in 4% PFA at room temperature for 20 min. Subsequently, cells were washed with PBS⁺ and permeabilised using 0.1% Triton X-100 (in PBS⁺). iPSC-CMs were blocked with 2.5% normal horse serum for 30 min. Primary antibodies targeting cTnT (mouse; 1:500; Abcam, ab8295), and/or MLC2v (rabbit; 1:500; Abcam, ab79935) were used in conjunction with DyLight anti-rabbit-488 and DyLight anti-mouse-594 secondary antibodies (Vector Laboratories, DK-8818). Cells were incubated with antibodies for one hour, with a wash in PBS⁺ separating the incubations. After washing with PBS⁺, cells were incubated in 1.2 μM DAPI for 5 min. Finally, coverslips were washed with PBS⁺, then briefly in H₂O, and mounted to microscope slides using ProLong Gold Antifade Mountant (Thermo Fisher; P10144). All steps were performed at room temperature on a rocking platform and slides were transferred to 4 °C once set.

Cells were fixed in 4% PFA at room temperature for 30 min and washed with PBS twice. Cells were then treated with 10% FBS, 5% BSA and 0.2% Triton X-100 in PBS for 15 min, allowing blocking and permeabilization. Incubation with various primary antibodies was overnight, 4 °C. Following primary antibody incubation and washes in 1X PBS, the cells were incubated with the Alexa-Fluor 488 or 594 secondary antibodies (1:400, Molecular Probes) and nuclei were stained with Dapi (1:5000, Calbiochem). Cells were visualized with a 10×/0.30 or 20×/0.40 (dry lens) objective using inverted microscopes (DMI4000 or THUNDER Imager 3D, Leica Microsystems). The images were captured with a digital camera (DFC365 FX, Leica Microsystems) using LAS-AF software (Leica Microsystems).
Antibodies used in this study are as following: cTNT (MA5-12960, Thermo Fisher Scientific), GATA4 (SC-25310 Santa Cruz), α-MHC (MF20) (14-6503-82, eBioscience), DDR2 (sc-81707, Santa Cruz), CD31 (e-ab-30820, Elabscience), MLC2v (ab79935, Abcam), α-Actinin (A7811, Merck).

For flow cytometry analysis of SSEA-4 or MYL2 expression, hESCs or cardiomyocytes were fixed with 4% paraformaldehyde (PFA) for 15 min at room temperature (RT). Cells were permeabilized with 0.3% PBSTr (Triton-X100) for 15 min, RT (this step was not needed for SSEA-4). Then cells were blocked with 3% bovine serum albumin (BSA) in PBS for 30 min. After that, the cells were incubated with anti-SSEA-4 antibody (Santa Cruz, sc59368, 1:200) or anti-MYL2 antibody (Abcam, ab79935, 1:100) at 4 °C overnight. The next day, the cells were washed with PBS three times and then incubated with fluorescence-conjugated secondary antibody for 1 h, RT. After washing with PBS three times, the cells were harvested for analysis.
To monitor cardiomyocyte differentiation, at different time points, the cells were dissociated into single cells and washed with PBS twice, then filtered with 40 μm strainer. The percentage of NKX2.5-eGFP⁺ cells was determined by flow cytometry. FlowJo 10 and NovoExpress software were used for data analysis.
For cell sorting, at differentiation day 7, the cells were dissociated and resuspended in RPMI/B27. After being filtered with 40 μm strainer, the NKX2.5-eGFP⁺ cells were sorted by Beckman Coulter MoFlo Astrios EQ for subsequent analysis.