Nanog

ESCs and EB outgrowths were fixed with 3% PFA in PBS at room temperature for 10 min and then were permeabilized with 0.5% Triton-X 100 (Sigma–Aldrich) in PBS at room temperature for 5 min. Nonspecific antibody binding was blocked by incubation in 3% bovine serum albumin (BSA; Sigma–Aldrich) in PBS at room temperature for 1 h. Next, primary antibodies were diluted with 0.5% BSA in PBS and incubated with cells at 4°C overnight. Primary antibodies against the following epitopes were used: Oct-4 (Santa Cruz Biotechnology; diluted 1:100), Nanog (Cosmo Bio Co.; diluted 1:200), myosin heavy chain (DSHB, diluted 1:10), MyoD (Santa Cruz Biotechnology; diluted 1:200), and cardiac Troponin T (Abcam; diluted 1:100). The next day, specimens were incubated with appropriate secondary antibodies conjugated with Alexa 488 (Molecular Probes) or Alexa 595 (Molecular Probes) diluted 1:200 and DRAQ5 diluted 1:1,000 in 0.5% BSA in PBS at room temperature for 2 h. Specimens were washed in PBS and mounted using the Fluorescent Mounting Medium (Dako). The specificity of primary antibodies was confirmed by incubating ESCs and EB outgrowths with secondary antibodies alone. Specimens were analyzed using an Axiovert 100M scanning confocal microscope (Zeiss) equipped with LSM 510 software. Figures were assembled using Adobe Photoshop CS6 Extended.

The cells growing on slides were fixed in 4% paraformaldehyde (PFA) for 30 min, and then permeabilized with 0.5% Triton™ X-100 for 15 min. Slides were blocked in 2.5% bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA) for 1 h and incubated in primary antibodies overnight at 4 °C, and subsequently incubated with secondary antibodies (AlexaFluor®, Invitrogen, Carlsbad, California, USA) for 1 h at room temperature. Imaging was performed using a Leica confocal microscope (Leica TCS SP8 MP, Chicago, IL). The primary antibodies included: SSEA-4 (1:500; Millipore, Billerica, MA, USA), TRA1-60 (1:500; Millipore, Billerica, MA, USA), TRA1-81 (1:500; Millipore, Billerica, MA, USA), Nanog (1:500; Cosmobio, Tokyo, Japan), OCT3/4 (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA), SOX2 (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA), TUJ1 (1:1000; Covance, Princeton, NJ, USA), HB9 (1:100; Developmental Studies Hybridoma Bank (DSHB), Iowa City, IA, USA), and ISL1/2 (1:200; DSHB, Iowa City, IA, USA).