Anti α fetoprotein

Immunostaining and alkaline phosphatase (AP) staining were performed as described previously [15 (link)]. Primary antibodies were anti-stage-specific embryonic antigen 1 (SSEA1, MC-480; Developmental Studies Hybridoma Bank, DSHB, Iowa City, IA), anti-Oct3/4 (MBL, Nagoya, Japan), anti-nestin (Rat-401; DSHB), anti-α-smooth muscle actin (Progen, Heidelberg, Germany), anti-neuronal class III β-tubulin (Tuj1; Covance, Richmond, CA), anti-Sox2 (Millipore, Bedford, MA), anti-glial fibrillary acidic protein (GFAP; Dako, Carpenteria, CA), anti-α-actinin (Sigma-Aldrich, Tokyo, Japan), anti-α-fetoprotein (R&D Systems, Minneapolis, MN), anti-Sox1 (Cell Signaling Tech, Beverly, MA), anti-Sox10 (Millipore), anti-PGP9.5 (Ultra Clone, Wellow, Isle of Wight, UK), and anti-GFP (Nakarai Tesque, Kyoto, Japan). Secondary antibodies were Alexa Fluor 568-conjugated goat anti-mouse IgG, Alexa Fluor 488-conjugated goat anti-mouse IgG1, Alexa Fluor 568-conjugated goat anti-mouse IgM, Alexa Fluor 647-conjugated goat anti-mouse IgG, Alexa Fluor 647-conjugated goat anti-rat IgG, and Alexa Fluor 488-conjugated goat anti-rabbit IgG (all purchased from Molecular Probes, Life Technologies, Eugene, OR).

Immunocytochemical analysis was performed as described previously [16 (link)–18 (link)]. The following primary antibodies were used in this study: anti-SSEA4 (1:300 dilution; Millipore), anti-TRA-1-60 (1:300 dilution; Millipore), anti-TRA-1-81 (1:300 dilution; Millipore), anti-Oct-3/4 (1:300 dilution; Santa Cruz Biotechnology), anti-human Nanog (1:800 dilution; Cell Signaling Technology), anti-Class IIIβ-tubulin (TUJ1; 1:500; Covance), anti-human smooth muscle actin (SMA; 1:50; DAKO), and anti-α-fetoprotein (1:100; R&D systems). Cells were incubated with a primary antibody diluted in 1% bovine serum albumin (BSA) and 5% serum containing PBS at 4°C overnight. Secondary staining was performed with an appropriate secondary antibody-conjugated to Alexa Fluor 488 or Alexa Fluor 594 (1:300; Life technologies) for 1 h at room temperature. Lectin staining was performed as described previously [19 (link),20 (link)]. Briefly, rBC2LCN (Wako) was fluorescent-labeled using FITC Labeling Kit-NH2 (Dojindo) according to the manufacturer’s instruction. Next, 10 μg/mL of FITC-conjugated rBC2LCN in 1% BSA containing PBS was used for staining of 4% paraformaldehyde-fixed cells for 1 h at room temperature. Cells were counterstained with DAPI (Dojindo). Images were collected with a BIOREVO BZ-9000 fluorescence microscope (Keyence).