Mrb435p

For immunofluorescence, cells or tissue sections were incubated with 0.2% Triton X-100 (Gibco) for 30 min and then were washed with PBS and incubated with blocking buffer composed of 3% BSA (Sigma-Aldrich, A9647) in PBS for 30 min at room temperature. The samples were then incubated with primary antibody overnight at 4 °C (SOX2 (Millipore, MAB5603, 1:400), PAX6 (RD, MAB1260, 1:1000); SOX1 (R&D, AF3369, 1: 400), NESTIN (Millipore, MAB5922, 1:400); MAP-2 (Millipore, MAB5622,1:600); GFAP (Sigma, G9269, 1:2000), DCX (Millipore, MABN707, 1:500), TUJ1 (Covance, MRB435P, 1:1000), NeuN (Millipore, ABN78,1:500), P-CREB (CST, 9198)). The following day, the samples were washed with PBS and incubated with the corresponding secondary antibody (Alexa Fluor 488 anti-mouse or 594 anti-rabbit (Invitrogen,1: 500)) for one hour at room temperature. Nuclei were counterstained with DAPI (Sigma-Aldrich, 32,670) for 15 min at room temperature. The cells were photographed using a confocal microscope (Carl Zeiss LSM710 Confocal). Tissue sections were photographed using a microscope (Leica, SP8) and the number of cells was counted using Image J software. The percentage of positive cells was calculated from the total nucleus population. All studies were performed for a minimum of 3 sections per sample, with 6 animals in each group.

Cells prepared for immunocytodetection experiments were cultured on poly-ornithine/laminin-coated round glass coverslips. Cells were fixed using 2 % paraformaldehyde for 10–15 min, washed twice with PBS, permeabilized using 0.1 % Triton X100 in PBS, and blocked using 0.5 % BSA in PBS. Primary antibodies used for microscopy included Oct3/4 (1:200; Santa Cruz Biotechnology C-10; sc-5279), Nanog (1:300; Novus Biologicals; NB100-58842), acetylated N-tubulin (clone 6-11B-1; 1:500; Sigma; T7451), neuronal class III β-tubulin (1:500; Covance; MRB-435P), Nestin (1:200; Millipore; MAB353), Pax6 (1:400; Covance; PRB-278P), and GFP (1:1000; Life Technologies; A-6455). Primary antibodies were incubated for 2 h at room temperature; cells were then washed three times with PBS (10 min each). Alexa Fluor 488 and Alexa Fluor 568 anti-mouse or anti-rabbit IgG conjugates (1:500; Molecular Probes; A-11001 and A-11011, respectively) were incubated for 1 h at room temperature in PBS containing 0.5 % BSA for primary antibody detection, followed by three PBS washes (10 min each). Nuclear staining was done with DAPI (2 μg/mL; Sigma).
The same protocol was applied on sections of embryoid bodies after fixation (2 h at 4 °C in 2 % PFA), dehydration (overnight at 4 °C in 30 % sucrose/PBS), inclusion in Tissue-Tek OCT compound (Sakura), and cryostat sectioning (12–16 μm) on SuperFrost glass slides (Thermo).

Primary antibodies used for Western blottings were as follows: rat anti-HA (Roche catalog 11-867-423), mouse anti-RPS5 (Abcam, ab58345), rabbit anti-RPS18 (Abcam, ab91293), rabbit anti-RPS15 (Abcam, ab90902), rabbit anti-RPL11 (Abcam, ab79352), rabbit anti-TUJ1 (Covance catalog MRB₄₃₅P), mouse anti-Rpl22 (Santa Cruz Biotechnology, sc-373993), rabbit anti-gERK (Sigma, M5670), and rabbit anti-albumin (Cedarlane, CLAG5140).
Primary antibodies used for immunostaining were as follows: rat anti-HA (Roche catalog 11-867-423), mouse Y10B (Abcam catalog ab37144), rabbit anti-TUJ1 (Covance catalog MRB₄₃₅P), rat anti-MBP (Millipore, MAB395-1ML), and chicken anti-NFH (Abcam, ab72996). Fluorescent secondary antibodies were from Jackson ImmunoResearch. Staining was observed using an Olympus FV1000 Confocal laser-scanning microscope at 40× or 60× magnification with oil-immersion Olympus UPLSAPO objective and was analyzed with FV10-ASW2.0 software.