Primary antibody against brdu

Detection of proliferating cells was performed following the methodology of Lee et al. (2012 (link)) with modifications. Adult (P75) male mice were injected peritoneally with BrdU (50 mg/ml) for 9 days, sacrificed the next day of the last injection, and immediately perfused. Subsequently, brain samples were processed for cryosectioning as described above. Coronal sections of 12‐μm thickness spanning the entire rostro‐caudal extent of the SCO were prepared. The frozen sections were washed in (TBS‐T) to remove excess OCT compound. Antigen retrieval was performed using 2 M HCl at 37°C for 10 min. Blocking was performed by incubation with 3% BSA in TBS‐T for 1 h at room temperature. Sections were incubated with a primary antibody against BrdU (1:100; AbD Serotec) overnight at 4°C. After washing with TBS‐T, the sections were incubated with a corresponding secondary antibody for 40 min at room temperature with counterstaining by 4′,6‐diamino‐2‐phenylindole (DAPI). Fluorescence images were obtained using a confocal microscope (ZEISS LSM800).

At approximately 32 months of age, mice were injected with BrdU for 8 days before sacrifice (50 mg/kg). Fixed hemibrains were sectioned coronally on a vibratome at 40 μm and then the brains were processed for BrdU and doublecortin (DCX) staining. Briefly, for BrdU staining sections were pretreated with 2N HCl at 37 °C, washed, and incubated overnight in primary antibody against BrdU (1:500; Abd Serotec). For DCX staining, sections were washed and incubated overnight in primary antibody against DCX (1:2500; Santa Cruz). Sections were then washed and incubated with biotinylated anti-rat secondary antibody for BrdU or anti-goat secondary antibody for DCX (Vector) for 1 hour and processed for diaminobenzidene staining using Vectastain ABC Elite kit (Vector). Stained sections were air-dried overnight, dehydrated, then coverslipped with Krystalon (EMD Millipore). Counting was done in the dentate gyrus (all layers) and quantified per unit area (mm²) for BrdU and per section for DCX.