Ab77013

POU5F1 (sc‐5279, Santa Cruz Biotechnology Inc., Santa Cruz, CA); NANOG (4903S, Cell Signaling Technology, Beverly, MA); SOX2 (3579, Cell Signaling); PAX6 (Developmental Studies Hybridoma Bank [DSHB], Iowa City, IA); TBXT (AF2085, R&D Systems Inc.); MESP1 (ab77013, Abcam, Cambridge, U.K.); ISL1 (39.4D5, DSHB); NKX2‐5 (sc‐14033, Santa Cruz Biotechnology Inc.); TNNT2 (MS‐295‐P1, Thermo Fisher Scientific); V5 (R960‐25, Thermo Fisher Scientific); SMAD4 (ab110175, Abcam; sc‐7966, Santa Cruz Biotechnology Inc.); GAPDH (8884S, Cell Signaling); CXCR4 (ab1670, Abcam); APLNR (702069, Thermo Fisher Scientific); CD13 (ab7417, Abcam).

The immunocytochemical staining were performed according to the protocol described previously [4 (link),38 (link)]. Briefly, cells seeded on slides or SIS patches were fixed with 4% paraformaldehyde (4% PFA) and permeabilized in 0.4% Triton X-100 (Sigma). After blocking with 10% normal goat serum (Vector Laboratories), the cells were then incubated with primary antibodies against octamer-binding transcription factor 4 (OCT4, Abcam, ab19857, 1:100), nanog homeobox (NANOG, Abcam, ab21624, 1:100), SSEA4 (Millipore, MAB4304,1:300), MESP1 (Abcam, ab77013, 1:100), ISL1 (Abcam, ab178400, 1:200), and NKX2-5 (Abcam, ab91196, 1:200), cTnT (Abcam, ab8295, 1:400), cTnI (Abcam, ab47003, 1:400), α-actinin (Abcam, ab9465, 1:400), or GATA4 (Stem Cell, SC-25310, 1:100) in 4 °C overnight and detected by DyLight 488- or 549-conjugated secondary antibodies. Nuclei were stained with Hoechst33258 (Sigma). Zeiss fluorescence microscope was used for slide observation and image capture.