Sheep polyclonal anti brdu

A series of coronal sections were selected to perform immunochemical staining of BrdU, nestin, DCX, NeuN, and laminin. The primary antibodies were applied including sheep polyclonal anti-BrdU (1 : 500, Abcam, USA), mouse monoclonal anti-BrdU (1 : 1000, Sigma-Aldrich, USA), mouse monoclonal anti-nestin (1 : 1000, Chemicon, USA), mouse monoclonal anti-neuronal nuclei (NeuN, 1 : 1000, Chemicon, USA), or rabbit polyclonal anti-laminin (1 : 200, Sigma-Aldrich, USA). After blocking with goat serum and incubation with primary antibodies overnight at 4°C, the slices were incubated with fluorescent-labeled secondary antibodies Alexa Fluor 488-conjugated anti-sheep IgG (1 : 200), Cy3-conjugated anti-mouse IgG (1 : 200), or Cy3-conjugated anti-rabbit IgG (1 : 200; all three antibodies from Jackson Immunoresearch Laboratories, USA) for 1 h at room temperature. For BrdU immunofluorescence, brain sections were pretreated for 30 min at 37°C with 2 N HCl and then rinsed for 10 min in 0.1 M boric acid (pH = 8.5) at room temperature followed by incubation with blocking solution. Phosphate-buffered saline instead of primary antibody was applied in negative control. The images were taken from four different fields at least (Olympus BX51; Olympus).

Animals were sacrificed by cervical dislocation, brains removed and fixed in 4% PFA for 2 weeks and then cryoprotected in 30% sucrose (w/v) in PBS for 48 hr, both at 4° C. Using a microtome, serial 30 μm coronal brain sections were obtained. Sections were stored at -20° C in cryoprotectant solution (ethylene glycol, glycerol, 0.1 M phosphate buffer, pH 7.4, 1:1:1:2 by volume). For immunohistological detection of BrdU, antigen retrieval was achieved with 1 M HCl at 37° C for 1 h. After washing in PBS, sections were blocked with a solution composed with PBS, 0.1% Triton X-100 and 1% bovine serum albumin (Sigma) for 1 hour. Primary antibodies were incubated overnight at 4° C. Sections were washed and incubated with fluorochrome-conjugated specific secondary antibodies overnight at 4° C. Slices were put on slides and mounted in Prolong Antifade Kit (Molecular Probes, Eugene, OR, USA). Confocal images were obtained with a Zeiss LSM 710 laser scanning confocal microscope using the Z-stack and tile functions as appropriate. Primary antibodies used were sheep polyclonal anti-BrdU (1:500, Abcam) and goat anti-doublecortin (DCX, 1:200, Santa Cruz, CA, USA).