Anti ssea 1

At the end of the differentiation period, the cells of each group were fixed with 4% paraformaldehyde for 30 min. Then, the cells were washed with 3x PBS. Next, the cells were blocked with 10% bovine serum albumin (BSA)-PBS at 37°C for 30 min and specific antigen (1:200 anti-Nanog, 1:200 anti-SSEA-1, 1:200 anti-Cvh, 1:250 anti-C-kit, 1:200 integrin α6, and 1:200 integrin β1; Millipore, Inc., Billerica, MA, USA) for one night at -4°C. Following a wash with 3x Phosphate Buffered Saline Tween (PBST), fluorescein isothiocyanate (FITC)- or PE-labeled goat anti-mouse immunoglobulin M (Bioss, China) was added and the incubation was continued at 37°C for 2 hours. The cells were then incubated with DAPI staining solution for 3 min at 37°C. After washing again with PBST, cells were observed under an inverted fluorescence microscope.

Immunostaining was carried out as previously described [6 (link)]. In brief, samples were fixed with 4% paraformaldehyde for 30 min at ~25 °C then washed three times with PBS. Non-specific sites were blocked with 500 μl of 2% bovine serum albumin (BSA) plus 0.45% Triton X-100 for 1 h at 20–25 °C. The samples were then incubated with the primary antibody overnight at 4 °C. Primary antibodies included anti-Oct4 (Santa Cruz, Dallas, TX, USA, 1:50), anti-Sox2 (Millipore, 1:200), anti-SSEA1 (Millipore, 1:200), and anti-Nanog (Abcam, Cambridge, UK, 1:100). The next day, samples were washed three times with PBS, incubated with an AlexaFlur 488-conjugated secondary antibody (diluted in 2% BSA plus 4.5% Triton X-100) at 25 °C for 1 h, and then imaged with a LSM780 Meta confocal microscope (Zeiss, Oberkochen, Germany). An alkaline phosphatase staining kit (Beyotime, Shanghai, China) was used to detect iPSC colonies according to the manufacturer’s instructions.