Anti neuronal class 3 β tubulin tuj1

Immunocytochemistry was performed as previously described10 (link). Following fixation with 4% paraformaldehyde, cells were sequentially incubated with 0.2‒0.5% Triton X-100, primary antibodies, and secondary antibodies. For diaminobenzidine staining, cells were incubated with the avidin–biotinylated horseradish peroxidase complex (Vector Laboratories, Burlingame, CA, USA) and reacted with a diaminobenzidine solution (Dojindo Laboratories, Kumamoto, Japan). For nuclear staining, cells were incubated with Hoechst 33258 (100 μg/mL). The following antibodies were commercially obtained: anti-TH from Merck Millipore, anti-MAP2 from Sigma-Aldrich, anti-pNF (SMI 31) and anti-neuronal class III β-tubulin (Tuj1) from Covance (Princeton, NJ, USA), anti-GFP from Nacalai Tesque, anti-GFAP from Merck Millipore, anti-integrin α5 (HMa5-1) and anti-integrin β1 (N-20) from Santa Cruz. Images of living or immunostained cells were obtained using an IX81 inverted research microscope (Olympus, Tokyo, Japan), a BZ-8100 fluorescence microscope (Keyence, Osaka, Japan), and an A1RMP multiphoton confocal microscope (Nikon, Tokyo, Japan). The fluorescence of immunostained cells was analyzed on a BD FACSAria III cell sorter (Becton-Dickinson).

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).