Mouse monoclonal antibody against brdu

Cervical cancer cells with stable knockdown and overexpression of PRDX1 or the corresponding control vector were seeded onto coverslips which were placed in the 6-well plate (1× 10⁴ cells/well) and bromodeoxyuridine (BrdU) was added 4 h before termination of cell culture. After that, the cells were fixed with 4% paraformaldehyde for 10 min at room temperature. Then endogenous peroxidase activity was inactivated with 0.3% hydrogen peroxide for 15 min at room temperature. RNase was added onto coverslips and incubated for 30 min at room temperature. The cells were then incubated with the mouse monoclonal antibody against BrdU (1:200, Cell Signaling Technology, Beverly, MA, USA) overnight at 4˚C. After washing with phosphate-buffered saline (PBS), the cells were labeled with Alexa Fluor 594-conjugated anti-mouse secondary antibodies (1:200, Thermo Fisher Scientific, Waltham, MA, USA). The nucleus was labeled with 4',6-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific, Waltham, MA, USA). Olympus fluorescence microscope was used to capture fluorescent images. The total numbers of DAPI positive cells and total numbers of BrdU positive cells were counted from at least five images from each sample. Each experiment was repeated three times.

Bromodeoxyuridine staining was used to detect newborn cells in the hippocampal DG and in the SN. For immunostaining the BrdU-labeled cells, the coronal sections containing the hippocampus and SN were rinsed (3 × 5 min each) with 0.05 M TB, incubated for 20 min at room temperature with 0.3% hydrogen peroxide, reacted with 0.5% Triton-100 for 15 min to increase permeability of cells, denatured by incubation for 30 min at 37°C with 2N HCl, and blocked by incubation for 1 h at room temperature in 100% normal goat serum dissolved in TB containing 0.5% Triton-100. They were then incubated overnight at 4°C with mouse monoclonal antibody against BrdU (1:100; Cell Signal, United States) and incubated sequentially for 1 h at room temperature with biotinylated horse anti-mouse IgG (H+L) antibodies (1:200; Vector, United States), 30 min at 37°C with streptavidin-horseradish peroxidase (1:300), and 40 s at room temperature with 3,3′-diaminobenzidine tetrachloride (DAB, Sigma, United States), then dehydrated in ethanol and xylene and coverslipped.