Fluorescence tagged secondary antibodies

The detected cells were washed three times with Dulbecco’s phosphate-buffered saline (DPBS), fixed for 30 min in 4% paraformaldehyde (PFA) at room temperature, then treated with 0.5% Triton X-100 for 10 min. Cells were incubated with primary antibodies against Oct4, Sox2, Nanog, and SSEA-1 (1:100, cell signaling technology, USA) at 4 °C overnight then incubated with fluorescence-tagged secondary antibodies (1:500, Santa Cruz, USA) at 37 °C for 2 h. After incubation, we stained with 4′,6-diamidino-2-phenylindole (DAPI) for 10 min after mounting and then observed and photographed samples under a confocal fluorescence microscope (A1R/A1, Nikon, Japan). For the alkaline phosphatase activity assay, we followed the instructions of the BCIP/NBT Alkaline Phosphatase Color Development Kit (Beyotime, China).

When the cultured cells reached 80-90% confluence, myogenic differentiation was induced by low serum medium comprised of DMEM supplemented with 2% horse serum (Hyclone Ltd) for 5 days. Myogenic cells and myotubes were identified by immunostaining, as described previously 37 (link). Myogenic cells were incubated with primary antibodies against MyoD (1:200; Santa Cruz, USA) and myotubes were incubated with primary antibodies against MHC (1:200; Cell Signaling Technology, USA) at 4℃ overnight, then incubated with fluorescence-tagged secondary antibodies (1:500; Santa Cruz, USA) at 37℃ for 1 h. After incubation and mounting, the samples were stained with 4',6-diamidino-2-phenylindole (DAPI) for 20 min. Samples were observed and photographed using a confocal fluorescence microscope (A1R/A1; Nikon, Japan).