5 bromo 2 deoxy uridine labeling and detection kit 1

After scraping the culture, astrocytes were fixed by 4% PFA at different time points, and incubated with primary antibodies (anti-GFAP antibody, 1:500; Dakocytomation; or anti-chondroitin sulfate antibody [CS-56], 1:200, Abcam) at 4°C overnight. Astrocytes were then rinsed with PBS, and incubated with a secondary antibody (FITC-goat anti-rabbit polyclonal, TRITC-goat anti-mouse polyclonal, Sigma) for 2 h at room temperature. Nuclei were counterstained with 4', 6-diamidino-2-phenylindole. To measure the proliferation of astrocytes, BrdU (1:1000) was added to the culture medium at the different time points after the scraping injury (12, 24, and 48 h). Procedures were performed according to instructions described in the 5-Bromo-2′-deoxy-uridine Labeling and Detection Kit I (Roche, USA). Representative sections were then observed with epifluorescence using a Leica microscope.

Cells were plated onto coverslips in 6-well culture plates in complete BEBM and cultured with or without growth factors for 48 and 72 h. BrdU was added 60 min prior to processing. Detection of BrdU incorporation was carried out according to manufacturer's instructions (5-Bromo-2′-deoxy-uridine Labeling and Detection Kit I, Roche Diagnostics) and visualized by microscopy (Axioplan 2, Zeiss). BrdU incorporation was measured by counting positive nuclei in a total of 500 cells in triplicate experiments.

BrdU (05650, Nacalai Tesuque, Kyoto, Japan) was IP injected into three-week-old CNP KO rats or WT rats at a dose of 50 μg/g body weight 2 h before being sacrificed. Target skeletal tissues were harvested, fixed overnight at 4°C in 4% paraformaldehyde solution, and decalcified for two weeks in 0.5 M EDTA. Decalcified samples were embedded in paraffin and sectioned. BrdU-positive cells were detected using a BrdU-specific antibody (5-Bromo-2´-deoxy-uridine Labeling and Detection Kit I, Roche Diagnostic GmBH, Germany). The number of BrdU-positive nuclei was determined per one slice of growth plate specimen of the proximal tibia.

The femoral arteries of mice were subjected to wire injury. After surgery, the mice were intraperitoneally injected with 1 mg Brdu and followed with infusion of Brdu using 7-day osmotic minipump (Alzet, Cupertino, CA, USA) at a rate of 60 µg/day for 7 days. The mice were killed and perfused with 4% PFA after killing and femoral artery was dissected. The femoral artery was embedding in paraffin and 5-μm sections were cut. After blocking and antigen retrieval, the slides were incubated with indicated antibodies. BrdU was stained with a 5-Bromo-2′-deoxy-Uridine Labeling and Detection Kit I (Roche, Indianapolis, IN, USA) following the kit instruction. Anti-SM22α antibody was obtained from Abcam (catalogue number: ab10135) with 1:200 dilution. Second antibody was DyLight 549 Streptavidin (Vector laboratories, Burlingame, CA, USA).

Cells were fixed in 3% paraformaldehyde EM grade (Electron Microscopy Sciences) for 15 min at RT. Cells were washed three times in PEM (100 mM Pipes, 1 mM EGTA, 1 mM MgSO4 pH = 6.9) and once in PEMS (PEM plus 1.2 M Sorbitol). Cells were treated with 0.5 mg/mL Zymolyase-100T (Seikagaku) for 1 h at 37°C. Cells were permeabilized in PEMS plus 0.5% Triton X-100 and incubated in ice for 10 min. Cells were then washed in PEM and incubated in PEMBAL (PEM + 1% BSA, 100 mM Lysine hydrochloride, 0.1% NaN3, pH = 6.9) containing 5 mM MgSO4 for 30 min. BrdU epitopes were exposed by incubating cells for 2 h at 37°C in PEMBAL containing 5 mM MgSO4, nucleases from the 5-Bromo-2′-deoxy-uridine labeling and detection kit I (Roche), and mouse anti-BrdU antibody (BD Biosciences, 347580). Cells were then washed three times with PEMBAL and incubated overnight with mouse anti-BrdU antibody (BD Biosciences, 347580). Cells were then washed three times in PEMBAL, incubated with Alexa Fluor 488 goat anti-mouse antibody (Invitrogen, A-11029) for 3 h, washed in PEMBAL, in PBS, and stained with DAPI. Alternatively, for heat denaturation, after permeabilization, cells were recovered in PEMS and incubated for 10 min at 95°C, followed by incubation in an ice/H2O bath for 5 min.