Nanog

The hiPS cells were cultured at the density of 1 × 10⁵ cells/cm² in a 24-well plate overnight and were then rinsed with PBS and fixed as described in “Alkaline phosphatase(AP) staining”. The cells were permeabilized with Perm/Wash buffer I (BD Phosflow; BD Biosciences) for 15 min. After three washes with 2% FBS in PBS, the cells were incubated with diluted primary antibodies overnight at 4 °C. The primary antibodies used were Octamer-binding transcription factor 3/4 (Oct3/4; 1:200; Santa Cruz Biotechnology, Dallas, TX, USA) and Nanog (1:200; ReproCELL, Yokohama, Japan). The cells were washed, and secondary antibodies (Goat polyclonal Ab to rabbit IgG-FITC(1:300, Santa Cruz, CA, USA) and Goat F(ab) anti-mouse IgG-FITC (1:300, Santa Cruz, CA USA)) were added to the cells for 1 h in the dark. The samples were then washed three times with 2% FBS in PBS, and one drop of mounting medium containing 4,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA, USA) was added. The cells were observed under a fluorescence microscope (Keyence, Osaka, Japan). Negative controls were prepared using the same procedure, without primary antibody treatment.

The iPSCs were seeded onto 12-well plates coated with Geltrex, grown to confluency, washed twice with PBS (−/−) and fixed with 10% formalin for 20 min at room temperature. The fixed cells were washed with PBS (−/−) then incubated with blocking buffer (10% FBS, 0.1% in PBS) for 30 min at room temperature to prevent any non-specific antibody binding. The primary antibodies were prepared by diluting with antibody buffer (0.2% FBS, 0.1% Triton X-100 in PBS stored at 4 °C). The following primary antibodies were used: Oct4 (1:100 dilution, Invitrogen), Sox2 (1:100 dilution, R&D Systems, USA) and Nanog (1:10 dilution, ReproCell, USA). After blocking, the wells were washed once with PBS (−/−) with 0.1% Triton X-100 and 300 µl of the primary antibody was added to each well. Primary antibodies were incubated overnight at 4 °C. After primary antibody incubation, the cells were washed (4 × 15 min) and the respective secondary antibodies were added to the cells and then incubated for 30 min at room temperature in the dark. Afterwards, the cells were washed (5 × 15 min) and stained with DAPI. All fluorescence images were taken on the spinning disk confocal (Quorum WaveFX Spinning Disc Confocal System). Secondary antibody controls were done to show no cross-reaction with the tissue (Figure S1).