Mouse monoclonal anti brdu antibody

The cell cycle was studied by using bromodeoxyuridine incorporation (BrdU; Sigma-Aldrich, USA). Briefly, cells were pulsed with BrdU at a final concentration of 10 μM for 15 min. Pulse-labeling experiments were performed by adding 10 μM BrdU to the medium during the last 30 min before analysis. After 30 min, the cells were harvested, washed once in PBS, fixed in 70% ethanol and stored at 4°C before analysis. Samples were then incubated with a mouse monoclonal anti-BrdU antibody (Roche Diagnostics, Milan, Italy) in complete medium containing 20% FCS and 0.06% Tween 20 (Calbiochem, San Diego, CA, USA) at room temperature for 1 h. After washing in PBS, cells were incubated with FITC-conjugated rabbit anti-mouse IgG 1:20 (DAKO, Glostrup, Denmark) in PBS for 1 h. Finally, cells were stained with a solution containing 5 μg/mL PI and 75 KU/mL RNase in PBS for 3 h, the top line of the cytograms represent BrdU-positive cells.

Sections (25 µm thick) were cut on a freezing microtome and processed for immunohistochemistry as previously described [20 (link)]. For BrdU detection, free-floating sections were pre-treated with 50% formamide, 0.3 M NaCl, 10 mM sodium citrate at 65 °C for 2 h, incubated in 2 M HCl at 40 °C for 1 h, and rinsed in 0.1 M borate buffer (pH 8.5) at room temperature for 10 min. Sections were incubated with the mouse monoclonal anti-BrdU antibody (1:300, Roche, Mannheim, Germany) at 4 °C for 24 h.

BrdU incorporation assays were performed as described previously^{61 (link)} using 10 μm BrdU and a mouse monoclonal anti-BrdU antibody (6 μg/ml; Roche), followed by a secondary IgG coupled to Alexa-488 (1:1000; Molecular Probes, Carlsbad, CA, USA). Slides were mounted with Mowial mounting medium and visualized by fluorescence microscopy using an Axiovert fluorescent microscope (Zeiss, Oberkochen, Germany). Data were obtained from three independent experiments.

Injected MSCs were detected by chicken polyclonal anti-GFP antibody (1 : 500, overnight at 4°C) (Abcam). Rat monoclonal CD45 (1 : 30, overnight at 4°C) (Novus Biological) was used to exclude the hematopoietic lineage in MSCs. Lung cells were identified by immunostaining for aquaporin 5 (AQP5; rabbit polyclonal, 1 : 100, overnight at 4°C) (Abcam) and surfactant protein C (SFTPC; rabbit polyclonal, 1 : 100, overnight at 4°C) (Santa Cruz Biotechnology). Cycling cells were visualized using mouse monoclonal anti-BrdU antibody (1 : 10, 1 h at 37°C) (Roche Diagnostics). The expression of hepatocyte growth factor (HGF; rabbit polyclonal, 1 : 100, overnight at 4°C) (Abcam) and its receptor c-Met (mouse monoclonal, 1 : 100, overnight at 4°C) (Cell Signaling) in the lung was also detected. Nuclei were stained with DAPI (Sigma-Aldrich). Secondary antibodies conjugated with FITC, TRITC, or Cy5 were used at the dilution of 1 : 100 for 1 h at 37°C (Jackson ImmunoResearch). The quantification of newly formed cells was performed by counting at least 200 AEC1 or AEC2 (n = 6 from each experimental group) and expressed as the percentage of BrdU-positive cells. Samples were analyzed with a Leica DM 5000B microscope a Zeiss LSM 700 confocal microscope.