Rabbit polyclonal anti nanog

Mouse iPSCs (SC201A-iPSC, passage 3, System Biosciences, Mountain View, CA, USA) were maintained in ESGRO complete plus clonal grade medium. The cells were passaged on reaching 80%–90% confluence at a ratio of 1:4 in 75 cm² flasks pre-coated with 0.1% gelatin. iPSCs of passages 11–13 were used for all experiments. The iPSCs were checked for SSEA-1, SOX2, Nanog and Oct4 expression by immunocytochemistry with the respective antibodies: mouse monoclonal anti-SSEA1-PE antibody (1:100, Millipore, Temecula, CA, USA), rabbit polyclonal anti-SOX2 (1:1000, Abcam, Cambridge, MA, USA), rabbit polyclonal anti-Nanog (1:700, Abcam) and rabbit polyclonal anti-Oct4 (1:1000, Abcam) at 4 °C overnight and fluorescein isothiocyanate-conjugated secondary antibody (1:100, Millipore) at room temperature for 1 h, images were captured by a fluorescence microscope (Eclipse E600, Nikon, Tokyo, Japan).

Cells were fixed in 4% PFA (Sigma), permeabilized with 0.5% Triton X-100 (Sigma) in DPBS (ThermoFisher), and blocked with 5% goat serum (ThermoFisher). All antibodies used in this study are detailed in Supplementary Table 9 (for example, primary antibodies used were rabbit anti-NANOG polyclonal (1:100, Abcam) and mouse anti-TRA-1-60 IgM (1:300, BD Biosciences)). Primary antibody incubation was conducted overnight at 4 °C on shakers followed by incubation with secondary antibodies (1:400) for 1 h. After labelling, cells were stained with 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI) (1:1,000, ThermoFisher) for 30 min. Images were taken using an IX71 inverted fluorescent microscope (Olympus). The following markers were assessed for respective differentiation assays: SOX17 and FOXA2 for endoderm progenitor differentiation experiments; SOX1 and PAX6 for neural differentiation experiments; PAX3 and PAX7 for skeletal muscle differentiation experiments; GATA6 and TTF1 for lung differentiation experiments.