5 bromo 2 deoxyuridine brdu

Four gerbils randomly selected from each group, were injected with 100 µg/kg body weight 5-bromo-2-deoxyuridine (BrdU; Roche Molecular Biochemicals) at the end of the experimental period. Six hours post-injection, the dissected pancreas was fixed in a 4% paraformaldehyde solution (pH 7.2) at room temperature for 16 h and embedded in paraffin blocks. Serial 5-μm paraffin-embedded tissue sections were mounted on slides, and they were rehydrated for the immunohistochemistry method.
Every sixth or seventh pancreas section was incubated with a guinea pig anti-insulin antibody. The anti-insulin antibody-stained areas of the pancreas were measured using anti-insulin (Zymed Laboratories, South San Francisco, CA, USA), and the percentage of the stained area from the total pancreas area was calculated. The β-cell proliferation was identified by incorporating BrdU into β-cells using the anti-BrdU antibody (Roche, Mannheim, Germany). The β-cell apoptosis was determined using a TUNEL assay (Roche) and double-stained with hematoxylin and eosin to visualize the islets [17 (link)]. The BrdU⁺ incorporated cells and apoptotic bodies of the islets were quantified based on the total islet numbers [27 (link)].

For cell proliferation studies, wounded animals were injected intraperitoneally with 5-bromo-2′-deoxyuridine (BrdU) at 0.25 mg/gram (Roche) three hours prior to the time that the animals were sacrificed. Wounds were excised, fixed, and processed for immunocytochemical analysis. Proliferating cells were detected using a BrdU labeling and detection kit (Roche, Indianapolis, IN, USA) according to the manufacturer′s instructions. For in vitro studies, sterile filtered 10 µM BrdU was prepared in media. After 12 h of vitamin D treatment, cells were washed with phosphate buffered saline (PBS), and replaced with the BrdU solution for a 2 h labeling period. Cells were fixed and then detected as above. Cell counting analysis was performed using the “spot” function on the Imaris (version 8.4, Bitplane, Concord, MA, USA) software.

BrdU crystals (5-Bromo-2-deoxyuridine, BrdU, Roche Applied Science, # 10280879001) were dissolved in 0.07 M NaOH solution warmed to 65°C. Pregnant mice (from gestational day 9 to 18) and male pups (from P0 to P8) were given a single i.p. injection around 10 a.m. (50mg/kg).
Tissue preparation. 21 to 23 days after birth (P21-P23), BrdU-injected male mice were anesthetized with isoflurane and perfused transcardially with a 0.9% NaCl solution, followed by an ice-cold solution of 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. Brains were quickly removed, postfixed in the same fixative for two hours at 4°C, and immersed in 20% sucrose in 0.1M phosphate-buffered saline (PBS) at 4°C overnight. Brains were finally either embedded in ice-cold OCT medium (optimal cutting temperature embedding medium, Tissue Tek, Sakura) and frozen in liquid nitrogen-cooled isopentane (most of the postnatally injected animals) or directly frozen in crushed dry ice without OCT (all prenatally injected animals). The different groups (n=3 to 7 per group) were then called “E9”, “E10”, “P1”, …: these developmental time points correspond to the day of the single i.p. BrdU injection.