Sox2 d6d9

HiPSCs were washed three times with PBS (Merck #L182-50) and fixed with 4% formaldehyde (Roth #P733.2) for 20 min at room temperature. After washing with PBS three times, cells were permeabilised with 0.5% Tween-20 (Roth #9127), 0.1% Triton X-100 (Roth 3051.3), 0.1% IGEPAL (Sigma #9036-19-5) in TBS for 20 min. Before blocking cells were washed with TBS three times and blocked with 5% donkey serum in TBS-T (0.1% Tween-20 in TBS) for 1 h (h). Afterwards washed once with TBS-T and incubated with the primary antibody OCT4 (rabbit-anti-OCT4 C30A3, Cell signaling Technology #2840 1:400), SOX2 (rabbit-anti-SOX2 D6D9, Cell signaling Technology #3579 1:400) or NANOG (rabbit-anti-NANOG D73G4, Cell signaling Technology #4903 1:200) respectively in 1% donkey serum in TBS-T at 4 °C overnight. Cells were washed three times with TBS-T for 5–10 min, and then incubated with secondary antibody (donkey-anti-rabbit Alexa488, Invitrogen #A21206, 1:400) in 1% donkey serum in TBS-T for 1 h at room temperature. The cells were washed three times with TBS-T for 5–10 min, then stained with 1:1000 dilution of Hoechst 33342 (Thermo Fisher #H3570) in TBS for 5 min, afterwards washed twice with TBS. Images were acquired with Cytation 1 (Biotek).