Anti ssea4

The cells were cultured on fibronectin coated cover slips for 24–48 hours. The cells were washed with PBS and incubated with anti-SSEA4 (Chemicon) antibody for 1 hour. The cells were washed and stained with fluorescent conjugated secondary antibody. The stained cells were microscopically documented.

The primary antibodies of anti-OCT4, anti-SSEA4, anti-NANOG, and anti-TRA-1-81 (all from Chemicon, USA) were used to characterize hESCs. Briefly, cells cultured on coverslips were fixed with 4% PFA at room temperature for 10 min, permeated with 0.1% Triton X-100 (Sigma, USA)/Phosphate Buffer Solution (PBS) on ice for 10 min, and blocked with fresh 2% bovine serum albumin (BSA: Sigma, USA)/PBS at room temperature for 30 min. The treated hESCs were washed with PBS for 5 min and then incubated with primary antibodies over night at 4°C. After rinsed with PBS for 5 min, the hESCs were stained by Cy2-conjugated or FITC-conjugated secondary antibodies (Jackson Immunoresearch, West Grove) in dark for 30 min. The hESCs were mounted with 4', 6-diamidino-2-phenylindole (DAPI: Vector Lab, USA) after being washed with PBS for 5 min, and then photographed under fluorescence microscope (Nikon, Japan).

Epithelial cells, oocyte-like cells, and colonies maintained on hAECs were fixed with 4% paraformaldehyde for 15 to 20 minutes at room temperature and permeabilized with 0.1% Triton X-100 for 10 minutes at room temperature. Cells were then blocked with blocking solution for 30 minutes and incubated with anti-OCT4 (rabbit anti-human 1:200; Santa Cruz), anti-NANOG (rabbit anti-human, 1:200; Chemicon, Rolling Meadows, IL, USA), anti-DAZL (goat anti human, 1:500; Santa Cruz), anti-STELLA (goat anti-human, 1:200; Santa Cruz), anti-ZPC (rabbit anti-human, 1:200; Santa Cruz), anti-SCP3 (rabbit anti-human, 1:800; Abcam), anti-GDF9 (rabbit anti-human, 1:200; Millipore), anti-SSEA4 (mouse anti-human, 1:100; Millipore), anti Tra-1-60 (mouse anti-human, 1:100; Millipore), and anti Tra-1-81(mouse anti-human, 1:100; Millipore) antibody for 1 hour at room temperature. Cells were then probed with fluorescein isothiocyanate-labeled IgG (1:200; Santa Cruz) or Rodamine (TRITC)-labeled IgG (1:100; Invitrogen) and incubated at room temperature for another 20 minutes. The slides were then covered with mounting medium (glycerol diluted 3:1 in PBS; Vector Laboratories, Burlingame, CA, USA). Fluorescence images were obtained with a Leica DMI3000 microscope.

Immunofluorescence staining was carried out according to standard protocols. Primary antibodies used were anti-OCT4 (#sc5279 Santa-Cruz, Heidelberg, Germany, 1:200), anti-NANOG (#sc293121 Santa-Cruz, 1:200), anti-TRA1-60 (MAB4360 Millipore, Darmstadt, Germany, 1:250) and anti-SSEA-4 (MAB4304 Millipore; 1:250). Secondary antibodies used were goat anti-mouse IgG Alexa-546 (#A-11030 Invitrogen, Schwerte, Germany) and goat anti-mouse IgM Alexa-488 (#A-21042 Invitrogen). Nuclei were co-stained with 4,6-diamidino-2-phenylindole DAPI (1:2000). Alkaline Phosphatase staining was performed using the Alkaline Phosphatase Staining Kit II (#00-0055 Stemgent, Glasgow, United Kingdom) according to the manufacturer’s instructions.

Karyotype analysis was performed by G-band analysis or Q-band analysis by Chromocenter (Tottori, Japan). Teratoma formation was assessed by injecting iPSCs (0.5–1.0 × 10⁶) into the testes of 8-week-old severe combined immunodeficient mice. Eight to ten weeks after iPSC transplantation, tumours were dissected and fixed with phosphate-buffered saline (PBS) containing 4% paraformaldehyde (Wako). Paraffin-embedded tissue was sectioned and stained with haematoxylin and eosin. For in vitro pluripotency assays, iPSCs were fixed in 4% paraformaldehyde/PBS for 15 min at room temperature and immunostained using the following primary antibodies: anti-OCT4 (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-SSEA4 (1:200, Millipore, Bedford, MA, USA), and DyLight-488-conjugated secondary antibodies (1:500, Thermo Fisher Scientific).

Anti-SSEA4, active β-catenin and total β-catenin monoclonal antibodies were purchased from Millipore (Billerica, MA, USA). Anti-Sca1-PE, CD34-PE, CD45-PE, CD73-PE, CD4-PerCP, CD8-FITC, CD25-APC, CD3ε and CD28 antibodies were purchased from BD Biosciences (San Jose, CA, USA). Anti-CD105-PE, CD178(FASL)-PE, Foxp3-PE, IL17-PE, and IFNγ-APC antibodies were purchased from eBioscience (San Diego, CA, USA). Anti-TERT, FASL and total β-catenin (ChIP grade) polyclonal antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-BRG1 antibody was purchased from Cell Signaling (Danvers, MA, USA). Anti-β-Actin antibody was purchased from Sigma-Aldrich (St. Louis, MO, USA).