Ab80892

Immunofluorescence staining was performed as described previously^{65 (link)}. In brief, pseudoblastocysts (96 or 120 hpi) or ESCs were fixed for 20 min at room temperature with 4% paraformaldehyde in PBS, washed with PBS containing 0.05% Tween 20 (PBST), and permeabilized for 30 min at room temperature with 0.4% Triton X-100 in PBS. They were then washed with PBST before incubation first for 1 h at room temperature with 1% BSA in PBST and then overnight at 4 °C with rabbit anti-CDX2 (1:1000 dilution, Abcam ab76541), mouse anti-OCT4 (1:500 for embryos and 1:250 for ESCs, Santa Cruz sc-5279), rabbit anti-NANOG (1:200, Abcam ab80892), mouse anti-GATA4 (1:200, Santa Cruz sc-25310; 1:1000, Santa Cruz sc-25310AF548), or mouse anti-DAB2 (1:1000, Santa Cruz sc-136964AF488) in the same solution. They were again washed with PBST, incubated for 1 h at room temperature with appropriate Alexa Fluor 488– or Alexa Fluor 568–conjugated secondary antibodies (Molecular Probes–Invitrogen), washed with PBST, and mounted in SlowFade Gold antifade reagent (Molecular Probes–Invitrogen) containing Hoechst 33342 (5 μg/ml) or RedDot2 (1:200, Biotium). Fluorescence images were acquired at multiple 1-μm intervals in the z-axis with the use of a confocal microscope (CV1000, Yokogawa).

Human iPSCs and iPSC‐derived neural cells were fixed with 4% paraformaldehyde (PFA) for 15 min at room temperature (RT). Prior to immunocytochemistry, cells were permeabilized with 0.1% Triton/phosphate‐buffered saline (PBS) for 15 min at RT and blocked with either 10% goat serum/PBS or 10% horse serum/PBS. Primary antibodies used were as follows: OCT4 (1:100; Santa Cruz, sc‐5279), NANOG (1:100; Abcam, AB80892), Tra‐1‐60 (1:100; Santa Cruz, sc‐21705), SSEA4 (1:100; BD Bioscience, 560796), α‐fetoprotein (1:150; R&D Systems, MAB1368), α‐smooth muscle actin (1:150; R&D Systems, MAB1420), Sox1 (1:100; R&D Systems, AF3369), Sox2 (1:100; R&D Systems, 245610), Nestin (1:500; Abcam, ab22035), Ki67 (1:100; BD Pharmingen, 550609), OTX2 (1:250; Millipore, AB9566), Pax6 (1:300; Biolegend, 901301), vimentin (1:100; Abcam, ab28028), vGLUT1 (1:2000; Synaptic systems, 135303), and β‐III tubulin (Tuj1) (1:150; R&D System, MAB1195). All primary antibodies were diluted in blocking solution and incubated overnight at 4°C, followed by washing in PBS 3 times for 15 min. Subsequently, appropriate Alexa Fluor 488 or 564 conjugated secondary antibodies were incubated for 1 h at RT. For nuclei staining, samples were incubated with DAPI (Sigma‐Aldrich) for 5 min and washed in PBS briefly. Finally, images were captured and analyzed using IN Cell 2200 (GE Healthcare).