Mouse anti brdu antibody

Cultured cells were fixed in a PBS solution containing 4% paraformaldehyde for 15 min and washed 3 times with PBS. A PBS solution containing 0.25% Triton was added to the cells for 15 min at room temperature and then incubated with 5% bovine serum albumin for 1 h. After removing this blocking reagent, cells were incubated in a humidified chamber at 4°C overnight with primary antibodies: mouse anti-BrdU antibody (1 : 1400; Cell Signaling Technology; Cat. No. #5292S), rabbit anti-NeuN antibody (1 : 50; Cell Signaling Technology; Cat. No. #24307S), and rabbit anti-NeuN antibody (1 : 50; Cell Signaling Technology; Cat. No. #24307S) diluted in blocking reagent. Then, cells were washed 3 times with PBS and incubated for 1 h in the dark at room temperature in the presence of the fluorescent secondary antibodies: goat anti-mouse IgG-FITC (1 : 100; Absin; Cat. No. #10) and goat anti-rabbit IgG/Cy3 (1 : 100; Absin; Cat. No. #AG04017512). Finally, the coverslips were mounted onto slides in PBS. The preparations were analysed under a fluorescent microscope (Olympus FV1200).

Microglial cultures were treated with each of the cytokines as described in Section “Stimulation of Primary Microglia,” after which 100 ng/mL BrdU was added to the medium. At 2–72 h later, microglia were fixed for 30 min in 4% PFA, blocked with 10% bovine serum albumin, then labeled overnight using the same goat anti-Iba1 antibody as in Section “Immunofluorescence” and mouse anti-BrdU antibody (1:400; Cell Signaling Technology, United States), followed by the anti-mouse and anti-goat secondary antibodies mentioned in Section “Immunofluorescence”. Finally, cells were stained for 5 min with DAPI (1:10,000) and imaged using a fluorescence microscope (Olympus BX51, Japan). Cells positive for BrdU and Iba1 were considered to be proliferating microglia.

MIN6B1 cells were seeded on poly-L-lysine-laminin-coated glass coverslips. BrdU (Roche) was added to the incubation medium for the last 6 h of culture. Thereafter, MIN6B1 cells were fixed in cold methanol and permeabilized using PBS supplemented with 0.5% saponin for 15 minutes. The coverslips were incubated in blocking buffer (PBS containing 0.5% saponin and 1% BSA) for 30 min and then sequentially exposed to a mouse anti-BrdU antibody (diluted 1/1400, Cell Signaling) for 1 h and to a goat anti-mouse Alexa Fluor 555 antibody (diluted 1/400, Invitrogen) for another 1 h. Finally, the cell nuclei were stained with Hoechst 33342 (1 μg/ml, Invitrogen) for 1 minute. Coverslips were mounted on microscope glass slides with Fluor-Save mounting medium (VWR International SA) and were visualized with a Zeiss Axiovision fluorescence microscope.

Cell proliferation was analyzed by BrdU incorporation assay. Briefly, cells were labeled with 40μM BrdU (Sigma Aldrich, St Louis, MO, USA) for 1h, fixed with 4% paraformaldehyde, and immunostained with a mouse anti-BrdU antibody (Cell Signaling Technology, Danvers, MA, USA) followed by staining with an anti-mouse secondary antibody conjugated to Alexa Fluor 488 (Cell Signaling Technology, Danvers, MA, USA) and counter-stained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI, Molecular Probes, Eugene, OR, USA). BrdU-positive cells were visualized and images were captured at 100× magnification using a fluorescent microscope (Olympus, Tokyo, Japan) and presented as the percentage of BrdU-positive nuclei over total number of nuclei counted. Cells were quantitated by randomly choosing at least four independent fields. At least 600 nuclei were counted.

For flow-cytometry-based detection of BrdU incorporation in 3T3NIH, serum-starved cell lines were incubated in RPMI culture medium containing 1× BrdU. After indicated time points, cells were detached and washed once in PBS. Cells were fixed and permeabilized with 70% ice cold ethanol for 5 mins at room temperature and rinsed two times in PBS followed by treatment with 1.5 M HCl at room temperature for 30 mins. Washed cells were stained for BrdU using Mouse anti-BrdU antibody (Cell Signaling, #5292) or mouse isotype control and for DNA using propidium iodide 10 μg/mL. Allophycocyanin-conjugated anti-mouse (Dianova, #115-136-146) was used as secondary antibody and measured in a flow cytometer.

Cell proliferation was explored using the BrdU incorporation assay. Glass coverslips in 24-well plates were inoculated with A549, A549-shLuc, or A549-shNMU cells and incubated for 24 hours. After that, the growth medium was replaced with a BrdU incorporation medium after 6 hours. The coverslips were incubated with mouse anti-BrdU antibody (Cat No.5292S, Cell signaling technology) overnight at 4°C. The coverslips were rinsed and incubated with Alex Flour 594-labeled anti-mouse IgG (Cat No. A-21203, ThermoFisher) for 1 hour at room temperature. Nuclei were labeled with DAPI. Images were captured using an Olympus fluorescence microscope (model: IX73, Tokyo, Japan). The numbers of BrdU-positive cells was counted from 5 random fields.