Anti brdu antibody

Perfused mouse brains were cut into 40 μm coronal sections using a cryostat. Sections were washed in PBS and then incubated in 1 M hydrochloric acid for 30 min at 47 °C, followed by further washes in PBS. Sections were then blocked in PBS containing 10 % goat serum and 0.3 % Triton X-100 for 2 h at room temperature and incubated overnight at 4 °C in anti-BrdU antibody (AbD Serotec) diluted 1:200 in blocking buffer. After washing in PBS containing 0.3 % Triton X-100 (PBST), sections were incubated in fluorescent anti-rat secondary antibody (1:300 in blocking buffer; Jackson ImmunoResearch) for 2 h at room temperature. Following further washes in PBST, sections were mounted onto slides using mounting medium containing bisbenzimide. Images were obtained by fluorescence microscopy and BrdU incorporation was quantified by counting of the number of BrdU-positive cells in every 12th section from each brain (sections were approximately 480 μm apart). The ratio between the total number of BrdU-positive cells in the supra- and infrapyramidal blades was calculated. Only cells within two cell diameters of the subgranular zone (SGZ; Kuhn et al. 1997 (link)) were included in cell counts.

Histological sections were blocked for 1 h at RT in a PBS containing 0.1% Triton-X (Sigma Aldrich) solution added with 5% normal donkey serum (NDS, Vector Labs) and incubation with specific antibodies against microglial/monocyte markers (Iba1; 1:500, Wako, CD68, F4/80 and CD11c; 1:100, AbD Serotec), neuroblasts (DCX; 1:1000, Millipore) and astroglial lineage cells (GFAP; 1:250, Sigma Aldrich) was performed overnight at 4°C. Proliferative cells were revealed by using an anti-BrdU antibody (1:100, AbD Serotec). To allow labeling of nuclear DNA, before blockage, sections were treated for 1 h with HCl 1M (RT) under agitation (Tang et al., 2007 (link)). Staining was revealed by 2-hour incubation period (RT) with appropriated secondary antibodies conjugated to Cy3 or Cy5 fluorophores (1:250, Jackson ImmunoResearch). DAPI (4′,6-Diamidino-2-phenylindole, 1:1000, Sigma Aldrich) was used for nuclear counterstaining and slides were mounted with ProLong Antifade (Life Technologies). Immunolabeled brain sections were analyzed and imaged using a confocal microscope (Olympus FluoView 500) with 40x (NA 1.30) and 60x oil-immersion (NA 1.25) objective lens (Olympus). Acquired images were adjusted for brightness and contrast using FIJI/ImageJ software.

Animals were injected with 50 mg/kg BrdU on 3 consecutive days and sacrificed after the indicated time. Sections were incubated with 2 M HCl in PBST (PBS+0.3% Triton) for 25 min at 37°C for denaturation of the DNA, neutralized with 0.1 M sodium tetraborate (pH 8.5) for 7 min at room temperature and stained with an anti-BrdU antibody (AbDSerotec). On day 107–109 at 12 a.m., the mice received a BrdU injection and at day 123, the animals were sacrificed. The brains were dissected and analyzed (Hillje et al., 2013 (link)). For quantification of BrdU+ cells in the DG, two sections each of 5 wt and 5 TRIM32 ko brains were analyzed, for quantification of BrdU+ cells in the OB, two sections of 4 wt and 4 TRIM32 ko mice were used. In each case, the mean of BrdU+ cells in TRIM32 ko tissue was normalized to the mean of BrdU+ cells in sections of wt brains.