Anti sox2

Chamber slides were pre-coated with poly-L-ornithine (Sigma Aldrich, USA) and 2x10⁴ single cells were seeded per well 4 hours before the actual experiments. To staining these cells, the cells were fixed with 4% paraformaldehyde (PFA) and incubated at room temperature. Then blocking solution (5% NHS and 0.5% Triton X-100 in PBS) was added at room temperature for 1 hour. And then the cells were stained with antibodies diluted in antibody dilution buffer (1% BSA and 0.3% Triton X-100 in PBS). The following antibodies were used as primary antibodies: anti-FoxM1 (Santa cruz, USA); anti-Sox2 (R&D Systems, Minneapolis, MN); anti-Nestin conjugated to Alexa-647 (BD Biosciences, San Jose, CA, USA); anti-pH2AX (Millipore, Billerica, MA); anti-GFAP (Millipore, Billerica, MA); anti-O4 (Millipore, Billerica, MA); anti-Tuj1 (Millipore, Billerica, MA), anti-NeuN (R&D Systems, Minneapolis, MN). Analysis was performed using confocal LSM 700 (Carl Zeiss, Germany).

First, free-floating sections were washed in PBS three times, followed by incubation in blocking buffer (0.1% Triton X-100, 1% bovine serum albumin, 5% normal goat serum in PBS) for 1 h. Then sections were incubated with primary antibody overnight at 4°C, washed for three times in PBS, and then incubated with secondary antibody for 2 h at 4°C. After 4′,6-diamidino-2-phenylindole (DAPI) treatment for 2 min, sections were washed again with PBS three times and mounted with Fluoromount-G (SouthernBiotech, Birmingham, AL). For BrdU incorporation, sections were treated before standard blocking protocol as follows: incubation with 2N HCl was performed at room temperature for 1 h, followed by soak in 0.1 m (pH 8.4) borate buffer for 10 min. After three washes in PBS, sections were processed according to standard protocol in blocking solution. The primary antibodies and their final concentrations used in the experiments were as follows: anti-BrdU (1:200, rat; AbD Serotec, Raleigh, NC), anti-pS6 (1:200, rabbit; Cell Signaling Technology, Danvers, MA), anti-GFP (1:1000, chicken; Abcam, Cambridge, MA), and anti-Sox2 (1:1000, goat; R&D Systems, Minneapolis, MN). Secondary antibodies were from Jackson ImmunoResearch Laboratories (West Grove, PA) and applied in a 1:1000 dilution.

Human NPCs were characterized by immunocytochemistry. Briefly, cells grown on glass coverslips were fixed with 4% paraformaldehyde and incubated in the blocking buffer (5% goat serum, 1% bovine serum albumin, and 0.1% Triton X-100) for 15 min. Cells were then incubated in primary antibodies diluted in the blocking buffer at 4 °C overnight. Appropriate secondary antibodies were used for single and double labeling. All secondary antibodies were tested for cross-reactivity and nonspecific immunoreactivity. The following primary antibodies were used, anti-SOX2 (1:200, R&D Systems), anti-Nestin (1:500, R&D Systems), anti-SOX1 (1:250, Millipore). Bis-benzamide (DAPI, 1:1000; Sigma) was used to visualize the nuclei. Images were captured using a Zeiss Axiovision microscope with z-stack split view function.