Mouse anti tra 1 60

iPSCs were fixed with 4% paraformaldehyde in PBS for 15 min and washed with PBS for 3 times, 5 min each. Cells were then permeabilized and blocked with 0.1% triton X and 2.5% bovine serum albumin in PBS for 1 hour and incubated in primary antibodies overnight at 4 °C (mouse anti-OCT4 from Santa Cruz (cat#SC-5279), 1:250; rabbit anti-LIN28 from Abcam (cat#AB46020), 1:500; mouse anti-SSEA4 from Abcam (cat#MC813), 1:200; rabbit anti-NANOG from GeneTex (cat#GTX100863), 1:300; goat anti-NANOG from R&D (cat#AF1997), 1:250; rabbit anti-H3K27me3 from Millipore (cat#07-449), 1:500); mouse anti-Tra-1-60 from Abcam (cat#AB16288), 1:500; and anti-Tra-1-81 Alexa647 from BD Biosciences (cat#BDB560124), 1:10). After washed with PBS for 3 times, 5 min each, cells were incubated in secondary antibodies if necessary (Alexa Fluor 488 donkey anti-mouse IgG (cat#A21202), 1:500; Alexa Fluor 555 donkey anti-rabbit IgG (cat#A31572), 1:500; Alexa Fluor 488 donkey anti-goat IgG (cat#A11055), 1:500, from Life Technologies) for 1 hour at room temperature and nuclei were stained using DAPI (1:5000).

hiPSCs growing for 5 days on 24 well plates were washed three times with DPBS (Life Technologies), fixed in PFA 4% for 20 min at RT, permeabilized with 0.1% Triton X-100 in PBS for 15 min at RT and blocked overnight at 4 °C in 4% BSA/PBS (Sigma Aldrich). The primary antibodies were incubated 1 h at RT, followed by incubation with the secondary antibodies. The following primary antibodies were used: goat anti-Oct3/4 (Santa Cruz Biotechnology Inc., Dallas, TX, USA), mouse anti-SSEA4 (Abcam, Cambridge, UK), mouse anti-TRA-1-60 (Abcam), mouse anti-KLF4 (Merck Millipore). Upon incubation with the appropriate secondary antibodies (AlexaFluor^® 488 Donkey anti-goat (Thermo Fisher Scientific, Whaltham, MA, USA), H&L (FITC) and Goat anti-Mouse, both from Abcam) cells were stained with DAPI (Life Technologies), fluorescence was observed in a Leica microscope and images were taken using LAS V3.8 software (Leica).

For pluripotency marker stainings, iPSC colonies were passaged as described above and grown on Matrigel^TM-coated coverslips in ES medium containing, 50% MEF-conditioned media (own preparation) supplemented with 5 ng/ml FGF2 (Sigma). Colonies were then stained for alkaline phosphatase according to the manufacturer’s protocol (Millipore) or were fixed with 4% paraformaldehyde (PFA) in PBS for 10 min at RT for analysis of pluripotency markers by immunofluorescence. Fixed colonies were incubated for 2 h in blocking solution (3% normal horse serum and 0.05–0.2% Triton-X100 in PBS). Plates were incubated over night at 4 °C using the following primary antibodies: rabbit anti-Nanog (1:500), rabbit anti-Oct4 (1:1000), mouse anti-SSEA4 (1:500), mouse anti TRA-1-60 (1:500) (all from Abcam) and mouse anti-Sox2 (1:500, R&D Systems). Differentiated EBs were stained with rabbit anti-α-SMA (1:500, Sigma Aldrich), mouse anti-α-Fetoprotein (1:500, Abcam), rabbit anti-GATA4 (1:500, Abcam), and mouse anti-TUJ1 (1:1000, Covance), mouse anti-Actinin (Sigma, 1:200) and mouse anti Beta-Catenin (BD Bioscience 1:500).