Ssea3

hESCs were fixed in 3.7% formaldehyde for 10 minutes at room temperature (RT). They were then incubated for >30 minutes in a gelatin block containing 1X PBS (Invitrogen), fish gelatin (Sigma), normal goat serum (NGS) (Jackson), normal donkey serum (NDS) (Jackson), 0.1% bovine serum albumin (BSA) (Sigma) and 0.25% Triton X. NGS was removed when staining with goat antibodies. Blocked cells were then incubated in primary antibodies diluted in gelatin block as follows: SOX2 (1:100, StemGent), AP2α (1:200, Developmental Studies Hybridoma Bank), non-muscle myosin IIA (1:1,000, Cell Signaling), phospho-myosin light chain (1:200, Cell Signaling), OCT4 (1:300, Millipore), SSEA-3 (1:200, Millipore), Nanog (1:600, Cell Signaling), Brachyury (1:300, R&D Systems). Primary antibodies were incubated for either 1 hour at RT or overnight at 4 °C. Alexa Fluor secondary antibodies (Invitrogen) were incubated at a dilution of 1:250 for 45 minutes at RT in the dark. Phalloidin conjugated to Alexa Fluor 594 was incubated for 20 minutes at RT (1:40 in gelatin block, Invitrogen). Finally cells were either incubated with DAPI (1:1,000 in PBS, Pierce) and mounted to slides with ProLong Gold (Invitrogen), or mounted in ProLong Gold with DAPI (Invitrogen).

iPSCs at passages 15–20 were grown to confluence in 48-well tissue culture plates on mouse feeders. Cells were fixed in 4% paraformaldehyde and permeabilized with 0.2% Triton X-100. Indirect immunofluorescence staining was then performed with antibodies against OCT3/4 (Abcam), SOX2 (R&D systems), SSEA3 (Millipore) and NANOG (Abcam), followed by Alexa fluorochrome-conjugated secondary antibodies (Life Technologies). Results were observed using Nikon Eclipse Ti-U inverted microscope with DS-Qi1 monochrome digital camera. The procedure used to stain for HNF4a and Albumin expression has been described in detail previously [28 (link)].

For immunostaining, the primary antibodies used included those against OCT3/4 (1:500, Santa Cruz), SOX2 (1:500, Santa Cruz), NANOG (1:500, Abcam), SSEA3 (1:50, Millipore), SSEA4 (1:50, Millipore), TRA-1–60 (1:50, Millipore), UTF1 (1: 200, Abcam), DPPA3 (1:50, Santa Cruz), ZSCAN4 (1:100, Millipore) and MBD3L2 (1:100, Abcam). The following secondary antibodies were used: Alexa Fluor 594-conjugated donkey anti-mouse IgG (1:500; Invitrogen), fluorescein isothiocyanate (FITC) 488-conjugated donkey anti-rabbit IgG (1:500; Invitrogen), and FITC 488-conjugated donkey anti-goat IgG (1:500; Invitrogen). Anti-H3K4me2 (Millipore), Anti-H3K4me3 (Millipore), Anti-H3K27me3 (Abcam) and Anti-H3K9me3 (Millipore) antibodies were used for ChIP experiments.

Immunofluorescence staining was conducted as described previously [23 (link)]. The primary antibodies used were OCT4 (1:200; Santa, Dallas, Texas, USA), SOX2 (1:200; Millipore), SSEA-1 (1:200; Millipore), SSEA-3 (1:200; Millipore), SSEA-4 (1:200; Millipore), TH1 (1:400; Millipore), TUJ1 (1:1000; Covance, Princeton, New Jersey, USA), NKX2.5 (1:200; Abcam, Cambridge, UK), and CTNT (1:200; R&D, Minneapolis, Minnesota, USA). The secondary antibodies were donkey anti-mouse IgG (H + L) conjugated to Cy3 (1:200; Jackson, Lansing, Michigan, USA), donkey anti-mouse IgG conjugated to Cy5 (1:200; Jackson), donkey anti-goat IgG conjugated to Cy5 (1:200; Jackson), donkey anti-rabbit IgG conjugated to fluorescein isothiocyanate (FITC) (1:200; Jackson), and donkey anti-rat IgG conjugated to Cy5 (1:200; Jackson).