Rat anti brdu antibody

To quantify proliferating cells, mice were injected intraperitoneally with the thymidine analog 5-bromo-2′-deoxyuridine (BrdU; 40 mg/kg body weight, 500 μl, SigmaAldrich) at 12 hours and 1 hour before sacrifice as described previously48 (link). Mice were euthanized and perfused through the left ventricle with saline, and skin were embedded in OCT compound and snap-frozen in liquid nitrogen. Sections were incubated with a rat anti-BrdU antibody (Abcam) to detect proliferating cells. For nuclear staining, BrdU-labeled tissue sections were counterstained with TO-PRO-3 (Invitrogen).

For the detection of newly-generated cells in the dentate gyrus, BrdU-specific immunofluorescence was performed, as previously described (5 (link)). The sections were first permeabilized by incubation with 0.5% Triton X-100 in PBS (1 ml/well) for 20 min at room temperature, and were then pretreated with 50% formamide-2X standard saline citrate (1 ml/well) at 65°C for 2 h, denatured in 2N HCl (2 ml/well) at 37°C for 30 min, and rinsed twice in 100 mM sodium borate (pH 8.5; 1 ml/well). For double labeling of BrdU and neuronal nuclear antigen (NeuN), the sections were incubated overnight with rat anti-BrdU antibody (1:300, Abcam, Biomeda, CA, USA) and mouse anti-NeuN antibody (1:500, Chemicon International, Temecula, CA, USA) at 4°C overnight following DNA denaturation. The sections were then incubated for 2 h with cy3-conjugated anti-rat secondary antibody for BrdU (1:200, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) and fluorescein isothiocyanate-conjugated anti-mouse secondary antibody for NeuN (1:200; Jackson ImmunoResearch Laboratories). The sections were then mounted on gelatin-coated glass slides, and the coverslips were mounted using fluorescent mounting medium (DakoCytomation, Carpinteria, CA, USA). Images of the fluorescent staining were captured using an epifluorescent microscope (Nikon Eclipse 50i; Nikon Inc., Melville, NY, USA).

Immunohistochemistry was conducted as Li et al. (2009) (link). Mice were anesthetized deeply with chloral hydrate (500 mg/kg, i.p.), transcardially perfused with ice-cold 0.9% NaCl and then 4% buffered formalin. Brains of mice that received intraperitoneal injections of BrdU were carefully and quickly removed and fixed in 4% paraformaldehyde at 22°C for 48 h for histochemistry of BrdU. Coronal 12-μm sections were cut and incubated free-floating for 24 h at 4°C in PBS containing both rat anti-BrdU antibody (1:200; Abcam, Cambridge, MA, United States) and mouse anti-NeuN antibody (1:1000; Chemicon, Temecula, CA, United States). After rinsing with PBS for three times, the sections were then incubated with Red-X-conjugated goat anti-rat IgG and FITC- conjugated goat anti-mouse IgG (1:200 for both; Jackson, MS, United States) to react to the corresponding primary antibody in PBS for 2 h at 22°C before mounting. The sections were photographed and analyzed by confocal microscope (Zeiss LSM510, Thornwood, NY, United States). The BrdU-positive cells were counted as described previously (Li et al., 2009 (link)). Briefly, the BrdU immunohistochemistry was performed for every sixth section throughout the entire hippocampus. All BrdU-positive cells in the hippocampus DG were counted by a blind observer and multiplied by 6, recorded as the total number of labeled cells in the DG.

To analyze BrdU-positive cells, 3 × 10⁵ MEFs, were plated in 5 plates/10 cm each (including coverslips), in DMEM supplemented with 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine and 10 mM Hepes. Cells were synchronized by serum deprivation (0.1% FBS) for 48 hours and later incubated with 10% FBS for 6, 12, 24, 30 and 48 hours, and 2 hours before harvesting, they were incubated with 10 µM BrdU. Cells were counted in a Neubauer chamber. Coverslips were washed 3 times with PBS, fixed using 4%PFA × 15 minutes, incubated with 2 M HCl for 15 minutes at 37 °C, blocked with 2% BSA-0.03% Triton x-100 during 1 hour and then incubated with a rat anti-BrdU antibody (Abcam). Finally, a goat anti-rat Alexa Fluor 488 labeled antibody (Invitrogen) was used and samples were analyzed using an epifluorescence microscope (Olympus IX81).

BrdU/NeuN-positive cells in the dentate gyrus were tested for immunofluorescence. In brief, the brain sections were permeabilized by incubation in 0.5% Triton X-100 in PBS for 20 min, then incubated in 50% formamide-2× standard saline citrate at 65 °C for 2 h, denaturated in 2 N HCl at 37 °C for 30 min, and rinsed twice in 100 mM sodium borate (pH 8.5). The sections were incubated overnight with a rat anti-BrdU antibody (1:500; Abcam, Cambridge, UK) and mouse anti-NeuN antibody (1:500; Millipore, Temecula, CA). The brain sections were then washed in PBS and incubated with the appropriate secondary antibodies for 1.5 h. The secondary antibodies used were anti-mouse IgG Alexa Fluor-488 and anti-rat IgG Alexa Fluor-550. Images were captured using an FV3000 confocal microscope (Olympus, Tokyo, Japan).

BrdU (50 mg/g of body weight) were injected intraperitoneally to mice 1 h before sacrifice. Dissected tumors were subjected to immunohistochemical staining. The primary antibody used was a rat anti-BrdU antibody (Abcam, Cambridge, UK) and the reaction was visualized using DAB. The number of cells positive for BrdU was counted on images captured under 400 × magnification in three fields from five tumors for each treatment group.