Mouse anti β tubulin

Cryostat sections (20 μm) were stained using standard immunohistochemistry. Primary antibodies: rabbit anti-GFAP (1:1000; Dako, Noble Park, VIC, Australia), mouse anti-GFAP (1:1000; Invitrogen, Mulgrave, VIC, Australia), rabbit antidoublecortin (DCX) (1:400; Cell Signaling, Arundel, Qld, Australia), rabbit anti-Pax6 (1:300; Covance), mouse antinestin (1:300; Cell Signaling), mouse anti-β-Tubulin (1:1000; Promega, Alexandria, NSW, Australia); mouse anti-BrdU (1:400; Roche, Hawthorn, VIC, Australia), rat anti-BrdU (1:200; Abcam, Cambridge, MA), mouse anti-HuC/D (1:250; Invitrogen), mouse anti-chondroitin sulfate proteoglycan (CSPG) (clone CS-56) (1:200; Sigma), rat anti mouse-CD11b (1:200; Invitrogen), and mouse anti-Sox2 (1:200, Sigma). Secondary antibodies: Alexa Fluor 488, 568, or 633; 1:1000 (Invitrogen). Nuclei were visualized with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma). Antigen retrieval was performed by incubation in 2-mol/L HCl for 15 min (BrdU) or 1-mol/L Tris-HCl (pH:8.0) at 90°C for 20 min (HuC/D).

Sections were labelled using standard immunohistochemical procedures to determine expression and localization of different proteins at the lesion site. Sections were post-fixed for 10 min in 4% PFA, followed by blocking solution (PBS-triton X containing 5% normal goat serum (Invitrogen, CA, USA)) for 1 h at room temperature. Antigen retrieval was performed by incubating the sections for 15 min in 2 M HCl prior to blocking for BrdU immunohistochemistry. Primary antibodies were diluted in blocking solution and sections were incubated overnight at 4 °C. After rinsing in PBS, sections were incubated for 2 h at room temperature with secondary antibodies diluted in blocking solution. Sections were mounted in Fluoromount (Dako, USA). Primary antibodies used were: mouse anti-NeuN (1:1000; Millipore); rabbit anti-pMAPK (mitogen-activated protein kinase 1:1000; Cell signalling); mouse anti-bromodeoxyuridine (1:400, Roche); rabbit anti-GFP (1:500; Invitrogen); mouse anti-β-tubulin (1:1000, Promega); rabbit anti-Ki67 (1:400, Thermo). Secondary antibodies used were: goat anti-rabbit or goat anti-mouse Alexa Fluor-488 or Alexa Fluor-594 (1:1000; Molecular Probes). Nuclei were visualised by staining with DAPI (4′,6-diamidino-2-phenylindole) (Sigma).