Anti pdgfrα

Cells were blocked with 1% BSA/PBS for 1 h at RT and incubated with anti-Iba-1 (1:300, BD Bioscience, San Jose, CA, USA), anti-NF-κB/p65 (1:200, Cell Signaling Technology, USA), anti-iNOS (1:300, Abcam, Burlingame, CA, USA), anti-Arg-1 (1:300, Gene Tex, Irvine, CA, USA), anti-GFAP (1:250, Thermo Fisher, USA), anti-BDNF (1:200, Novusbio, Centennial, CO, USA), anti-GDNF (1:500, Abcam, USA), anti-PDGFRα (1:300, Millipore, Germany) and anti-Ki67 (1:200, BD Pharmingen, USA) at 4°C for overnight, followed by corresponding secondary antibodies at RT for 1 h. The results were repeated three times with similar results. Analysis and quantification were done by Image-Pro Plus 6.0 software.

Immunohistochemistry was performed as previously described [44] (link). Briefly, injected animals were perfused at different time points with 4% paraformaldehyde (PFA). The brains were removed and immersed in 4% PFA overnight at 4°C and then equilibrated in 30% sucrose. Entire brains were processed in sagittal 40 microns microtome sections. Sections which were positive with GFP-labelled cells were blocked with 5% donkey serum, permeabilized with 0.25% Triton X in TBS for 30 minutes before application of primary antibodies of rabbit anti-GFAP (Dako, Glostrup, Denmark), anti-PDGFRα (Millipore), goat anti-doublecortin (Santa Cruz, CA, USA), mouse anti-human nestin (Millipore) and anti-human nuclei (Millipore) at dilutions of 1∶100 to 1∶500 for overnight incubation at 4°C. Incubations with secondary antibody (1∶500) of 647 donkey anti-rabbit or 647 donkey anti-goat with 555 donkey anti-mouse for one hour at room temperature were performed, followed by a five minute staining with DAPI (Millipore), before sections were mounted on slides with mounting medium. The staining was viewed using Zeiss LSM 710 confocal system (Carl Zeiss Pte Ltd, Singapore) at 63X magnification.

Mice brains were cut into several brain slices (25 µm) by microtome. At room temperature, the brain slides were sealed with 1% immune tissue blocking fluid for 2 h and incubated overnight with rabbit anti-MBP (1:100, Abcam, Cambridge, UK), anti-Iba-1 (1:100, Wako, Japan), anti-CC1 (1:100, Millipore, Burlington, MA, USA), goat anti-Olig2 (1:100, Millipore, Burlington, MA, USA), anti-PDGFRα (1:100, Millipore, Burlington, MA, USA) respectively at 4 °C. Subsequently, the brain slides were incubated with the corresponding secondary antibody at room temperature for 1 h. Then, the corpus callosum site of slices was photographed with a multiphoton laser scanning confocal microscopy system (Olympus Fluoview FV1000) or common fluorescence microscope (Leica DMI6000).