Anti nanog

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.

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).

Cells were lysed in lysis buffer (50 mM Tris pH 8, 120 mM NaCl, 0.5% NP-40) supplemented with 2% protease inhibitors (Aprotinin, Leupeptin 20 μg/mL, sodium orthovanadate 1 mM and PMSF 0.1 mg/mL, all from Sigma-Aldrich, St. Louis, MO, USA). Proteins were separated by SDS-PAGE under reducing conditions and transferred onto nitrocellulose membranes using a Bio-Rad wet transfer system. The membranes were then blocked with 5% nonfat dry milk and 1% NGS in TBS-T (Tris buffer saline–0.05% Tween), followed by overnight incubation at 4 °C with the corresponding specific primary antibodies: rabbit polyclonal anti-Nanog (1:2000, #AB5731, Sigma-Aldrich, St. Louis, MO, USA); mouse monoclonal (C-10) anti-Oct-3/4 (1:1000, sc-5279 Santa Cruz Biotechnology, Dallas, TX, USA); mouse monoclonal (6C5) anti-GAPDH (loading control; 1:50,000, ab8245, Abcam, Cambridge, United Kingdom,). After incubation, membranes were washed in TBS-T and HRP-conjugated secondary antibodies were added for 45 min at RT (ThermoFisher Scientific, Waltham, MA, USA #A31460 and #A31430, 1:10,000). HRP-conjugated proteins were detected with Super Signal West-Pico Chemiluminescent Substrate (Pierce, ThermoFisher Scientific, Waltham, MA, USA).