Rabbit antidoublecortin dcx

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).

Mice were anesthetized with ketamine (80 mg/kg, i.p.; Imalgène) and xylazine (20 mg/kg, i.p.; Rompun, Bayer), transcardially perfused with 4% paraformaldehyde (PFA), and cryopreserved in 30% sucrose. Brains were horizontally cut in 40-µm-thick slices in a cryotome (Microm HM450). Slices were rinsed with PBS and blocked in 10% BSA and 0.3% Triton for 2 h at room temperature. Slices were incubated with the following primary antibodies overnight at 4°C: chicken anti-green fluorescent protein (1:1000; Millipore); rabbit anti-doublecortin (DCX; 1:2000; Cell Signaling Technology); and mouse anti-NeuN (1:250; Millipore). Slices were incubated with the respective secondary antibodies for 3.5 h at room temperature, as follows: donkey anti-chicken IgG Cy2 (1:500); donkey anti-rabbit IgG Cy3 (1:600); and Alexa Fluor 647 donkey anti-mouse (1:400; all from Jackson ImmunoResearch). Slices were mounted in Fluoromount-G (Southern Biotech; Cabezas et al., 2013 (link)). Images were acquired on an inverted confocal microscope (SP2, Leica) and were obtained from stacks of 10–17 sections, 2.5 µm apart, using a 63×/1.32 numerical aperture objective. Images were acquired from four slices per animal of five saline- or METH-treated mice. Cell counts were calculated using ImageJ software. Results are expressed as the percentage of total GFP cells expressing DCX and/or NeuN.