Anti nanog ab5731

Western blotting was performed as described previously52 (link). Briefly, protein lysates were prepared in RIPA buffer supplemented with complete protease inhibitor (Roche Applied Science). Proteins were then separated on 6–15% SDS-PAGE, transferred onto PVDF membranes, and probed with anti-CIBZ [amino acids 1184–1197 (EQKDDIKAFAENVL) of CIBZ]17 (link), anti-Oct3/4 (MAB1759, R&D Systems), anti-Sox2 (S1451, Sigma-Aldrich), anti-Nanog (AB5731, Millipore), anti-α-tubulin (clone DM 1A, Sigma-Aldrich), anti-Brachyury (sc-17743, Santa Cruz Biotechnology), anti-Nkx-2.5 (sc-8697, Santa Cruz Biotechnology), anti-Islet 1 (ab109517, Abcam), anti-cTnI (ab19615, Abcam), and anti-MHC (clone MF20, Developmental Studies Hybridoma Bank) antibodies. HRP-conjugated anti-mouse or anti-rabbit IgG (Cell Signaling) were used as secondary antibodies.

ES cells on coverslips were fixed in 4% paraformaldehyde/ phosphate buffer saline (PBS) for 15 min at room temperature. The fixed cells were then permeabilized with 0.5% Triton X-100 for 10 min and were blocked in 3% bovine serum albumin for 1 h at room temperature. The cells were incubated overnight at 4 °C with primary antibodies, anti-oct3/4 (sc-5279; Santa Cruz, Dallas, TX, USA), anti-sox2 (sc-17320; Santa Cruz), anti-nanog (AB5731; Millipore, Billerica, MA, USA), and anti-SSEA1 (sc-21702; Santa Cruz). The cells were treated with the secondary antibodies for 1 h at room temperature. The nuclei were dyed with 4′,6-diamidino-2-phenylindole (DAPI). Images were acquired by the fluorescent microscope (Zeiss, Oberkochen, Germany).

Cultured cells and frozen tissue sections were fixed with 4% paraformaldehyde and then stained with the following primary antibodies: anti-Sox10 (N-20; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-PDGF receptor α-chain (558774; BD Pharmingen, San Diego, CA, USA), anti-PDGF receptor β-chain (#3169; Cell Signaling Technology, Danvers, MA, USA), anti-GFP (ab13970; Abcam, Cambridge, UK), anti-Ap2α, anti-AP-2β (Cell Signaling Technology), anti-Nanog (AB5731; Millipore, Temecula, CA, USA) anti-Nestin (G-20; Santa Cruz Biotechnology), anti-Oct3/4 (MAB1759; R&D Systems, Minneapolis, MN, USA), anti-PAR4 (3G9H7; Santa Cruz Biotechnology), anti-Sox2 (Y-17; Santa Cruz Biotechnology), anti-alpha smooth muscle actin (ab32575; Abcam), anti-βIII-tubulin (MAB1195; R&D Systems), anti-glial fibrillary acidic protein (MAB360; Chemicon International, Temecula, CA, USA) and anti-GFP (MBL, Nagoya, Japan). After washing with Tris-buffered saline, the sections were stained with Alexa Fluor-488 or Alexa Fluor-568 conjugated secondary antibodies (Invitrogen). All sections were counterstained with Hoechst 33342 (Invitrogen).