Rat anti pdgfrα

Animals were sacrificed, and serial sections (18-24 μm in thickness) were prepared. Sections were blocked by PBS containing 0.3% Triton X-100 and 5% bovine serum albumin (BSA) for 1.5 h. Primary antibodies were incubated overnight as the followings: rabbit anti-NG2 antibody (1 : 200, Millipore), rabbit anti-CNPase (1 : 200, Millipore), rabbit anti-MBP antibody (1 : 200, GeneTex), rabbit anti-Ki67 antibody (1 : 200, GeneTex), rat anti-PDGFRα(1 : 500, Abcam), rabbit anticleaved caspase-3 (1 : 200, GeneTex), and goat anti-Sox10 antibody (1 : 100, Santa Cruz Biotech). After washing, sections were incubated with secondary antibodies conjugated with Alexa Fluor 488 (donkey anti-rabbit, 1 : 400, Molecular probes) or Alexa Fluor 594 (donkey anti-goat or anti-rat IgG, 1 : 800, Molecular probes) for 2-4 h at room temperature. The nuclei were stained by DAPI (1 : 1500, Sigma). Images were taken by using confocal microscope (FV3000, Olympus) with same setting to reduce variation.

The corpus callosum was dissected and homogenized in an ice-cold lysis buffer. Western blot was carried out by standard protocol with proper antibodies like mouse anti-glyceraldehyde-3-hposphate dehydrogenase (anti-GAPDH; 1:5000; Kang-chen, China), rat anti-PDGFRα (1:500; Abcam, USA ); rabbit anti-Olig2 (1:500; Millipore, USA); rabbit anti-NG2 (1:500; Millipore, USA) or rat anti-MBP (1:500; Millipore, USA) overnight at 4 °C. Membranes were washed three times with TBST buffer and incubated with IRDye 800 anti-rabbit Molecular Probe (1:8000; LI-COR Biosciences, USA) or IRDye 700 anti-mouse/rat Molecular Probe (1:3000; LI-COR Biosciences, USA) for 2 h at room temperature. Images were acquired with the Odyssey infrared imaging system and analyzed by the Odyssey software49 (link).