Mouse anti ascl1 antibody

Each animal was deeply anesthetized by intraperitoneal injection of pentobarbital (20 mg/kg), and then perfused with chilled phosphate-buffered saline, followed by 4% paraformaldehyde in 0.1 mol/l phosphate buffer. After post-fixation overnight, 50 µm thick sections were cut with a vibrating blade microtome (VT1000S; Leica, Wetzlar, Germany). For immunohistochemistry, the following primary antibodies were used: rabbit anti-GFP antibody (1:500, MBL, Nagoya, Japan), goat anti-GFP antibody (1:200, Abcam, Cambridge, UK), rabbit anti-Iba1 antibody (1:500, Wako, Osaka, Japan); rabbit anti-glial fibrillary acidic protein (GFAP) antibody (1:500, Dako, Glostrup, Denmark); goat anti-PDGFRα antibody (1:100, R&D Systems, MN, U.S.A); rabbit anti-GST-π antibody (1:500, Enzo life sciences, NY, U.S.A.); rabbit anti-nestin antibody (1:200, Santa Cruz Biotechnology, CA, U.S.A.); goat anti-doublecortin (Dcx) antibody (1:100, Santa Cruz Biotechnology); mouse anti-betaIII tubulin (Tuj1) antibody (1:100, Santa Cruz Biotechnology); mouse anti-NeuN antibody (1:100, Millipore, MA, U.S.A.); mouse anti-Ascl1 antibody (1:100, BD Pharmingen, NJ, U.S.A.); rabbit anti-Sox2 antibody (1:100, Santa Cruz Biotechnology); mouse anti-NeuroD1 antibody (1:100, Abcam). Each primary antibody was detected by appropriate secondary antibodies conjugated with Alexa Fluor 488 or 555^TM (Molecular Probes, OR, U.S.A.).