Rabbit anti map 2

Neurospheres or cells adhered to PO-precoated coverslips were incubated in 4% paraformaldehyde for 10 min at room temperature and then washed with phosphate buffer saline (PBS, pH ~7.4) for three times. The samples were permeated with 0.5% Triton X-100 PBS for 30 min and blocked by 5% bovine serum albumin (BSA) for 2 h after washing three times. Thereafter, the samples were incubated in primary antibodies, goat anti-Nestin (1 : 100, Santa Cruz Biotechnology, CA, USA), rabbit anti-MAP-2 (1 : 100, Proteintech Group, Inc., Beijing, China), rabbit anti-glial fibrillary acidic protein (GFAP, 1 : 100, Abcam, Cambridge, UK), mouse anti-Olig2 (1 : 100, Milipore Corp., Billerica, MA, USA), rabbit anti-α-Smooth Muscle Actin (ACTA2) (1 : 100, Beyotime, Beijing, China), and mouse anti-Tubulin (1 : 100, Beyotime, Beijing, China) for 10–14 h at 4°C. After washing, relative fluorescence secondary antibodies were incubated at room temperature for 2 hours. Cell nuclei were counterstained with 4′-6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, St. Louis, MO) for 10 min at room temperature. Then, coverslips were mounted onto glass slides and the images were captured by a confocal microscope (Carl Zeiss, LSM780, Weimar, Germany) and examined using Zen 2011 software (Carl Zeiss, Weimar, Germany).

The 30 μm coronal cryosections were applied to glass slides and allowed to dry at room temperature overnight. Tissue sections were washed in phosphate-buffered saline (PBS) for 15 min and then blocked in 5% donkey serum, 1% BSA, and 0.2% Triton X-100 for 1 h at room temperature and incubated with primary antibody at 4 °C overnight. Mouse anti-pY99 (1:1000, Santa Cruz), rabbit anti-MAP2 (1:200, Protein Tech Co.), rabbit anti-pY416 (1:200, Cell Signaling), rabbit anti-pan Src antibody (1:200, Santa Cruz), rabbit anti-Dcx (1:200, [17 (link)], rabbit anti-Tbr1 (1:200, Abcam), rabbit anti-GAD67 (1:200, Millipore), and mouse anti-GM130 (1:200, BD) were used as primary antibodies. Donkey anti-species IgG conjugated with Alexa 488, Alexa 555 or Alexa 647, and Alexa 555-conjugated phalloidin were used as secondary antibodies. Hoechst 33342 (2 μg/ml, Molecular Probes) was used to visualize individual cell nuclei. Images were collected with a Zeiss LSM780 laser scanning confocal microscope (Advanced Fluorescence Imaging Core, SUNY Upstate Medical University). To compare the tyrosine phosphorylation response after different treatment, the average pY99 signal intensity was collected and normalized to the MAP2 immunosignal intensity and then compared among different treated group.