5 bromo 2 deoxyuridine brdu

Perfusion, dissection, and immunofluorescence staining were conducted according to standard protocols as previously described [17 (link)]. The following are the antibodies used: mouse anti-BrdU (1:50 dilution; BD Pharmingen, Franklin Lakes, NJ, USA), rabbit anti-Cux1 (1:100 dilution; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-Phospho-Histone H3 (1:250 dilution; Millipore, Billerica, MA, USA), rabbit anti-Pax 6 (1:500 dilution; Covance, Princeton, NJ, USA), rabbit anti-Tbr2 (1:500 dilution; Abcam, Cambridge, UK), mouse anti-Tuj1 (1:500 dilution; Covance, Princeton, NJ, USA), rabbit anti-Gli3 (1:100 dilution; Santa Cruz Biotechnology, Dallas, TX, USA), and rabbit anti-Cleaved Caspase 3 (1:300 dilution; Cell Signaling, Madison, WI, USA).
For 5-bromo-2-deoxyuridine (BrdU, Sigma, St. Louis, MO, USA) labeling, pregnant dams were treated with 50 μg/g BrdU by intraperitoneal injection for 4 h prior to dissection at E16.5. DiI labeling was conducted by placing small crystals of the lipophilic tracer (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine; Invitrogen, Waltham, MA, USA) in the neocortex to target the upper layer (2/3) and then remained in 4% paraformaldehyde (PFA). After 6 weeks, brains were sectioned at 100 μm, counterstained with bisbenzimide, mounted, and imaged.

Eight naturally-delivered male pups from six pregnant ferrets, which were purchased from Japan SLC (Hamamatsu, Japan), were used in the experiments. The pups were reared with lactating dams (3–5 pups/mother) in stainless steel cages (80 cm × 50 cm × 35 cm) maintained at 21.5 ± 2.5 °C under 12-h artificial illumination in the Facility of Animal Breeding, Nakaizu Laboratory Japan SLC (Izu, Japan). All lactating dams were fed a pellet feed (High-Density Ferret Diet 5L14, PMI Feeds, Inc., St. Louis, MO, USA) and ad libitum tap water.
The thymidine analogs and VPA were administered on a schedule designed according to a previous study [10 (link)]. Eight male pups were intraperitoneally injected with 30 μg/g body weight of 5-ethynyl-2′-deoxyuridine (EdU) (Sigma-Aldrich, St. Louis, MO, USA) at PD 5 and 30 μg/g body weight of 5-bromo-2′-deoxyuridine (BrdU) (Sigma-Aldrich) at PD 7, respectively. Four pups were intraperitoneally administered 200 µg/g body weight of VPA on PDs 6 and 7. Four unexposed pups were used as controls. VPA was injected a second time at the same time as the BrdU injection. On PD 20, all animals were perfused with 4% paraformaldehyde (PFA) (Merck, Darmstadt, Germany)-phosphate buffered saline (PBS) (pH 7.4) under deep anesthesia with ~2% isoflurane gas. The cerebellum was removed and immersed in the same fixative.

Salmon calcitonin (sCT, H-2260; Bachem), pramlintide (SC-476040; Santa Cruz) and liraglutide (gift of NovoNordisk, Denmark) were dissolved in 0.1 M PBS (pH 7.4) for systemic (IP) injections. Amylin (H-9475.100; Bachem), GLP-1 (7–36; H-67951000, Bachem) and Angiotensin II (Ang II: Bachem) were dissolved in artificial cerebrospinal fluid (aCFS; Harvard Apparatus) for central injection. 5′-bromo-2′-deoxyuridine (BrdU; Sigma) was dissolved in double-distilled water and heated to 40–50 °C.

A single intraperitoneal injection of 50 µg/g body weight 5-bromo-2-deoxyuridine (BrdU; Sigma-Aldrich, Cat. B5002) in physiological saline was administered 1 h prior to perfusion fixation in 4% PFA.

Chromosome-orientation fluorescence in situ hybridization (CO-FISH) was employed to evaluate IR-induced chromosomal instability and performed as previously described (62 (link), 70 (link)) with some modification. Following irradiation, cell cultures were incubated for various times, trypsinized, and subcultured into medium containing 5-bromo-2-deoxyuridine (BrdU, 10 μM; Sigma-Aldrich) for one cell cycle. Slides were stained with Hoechst 33258 (0.50 ng/μL; Sigma-Aldrich) for 15 min and exposed to 365 nm UV light (Stratalinker 2400) for 25 min. Following UV exposure, BrdU incorporated strands were digested with Exonuclease III (3 U/μL in provided reaction buffer; Promega) at room temperature for 10 min. Slides were hybridized with a Cy-3 conjugated (TTAGGG)₃ PNA telomere probe (0.2 μg/mL; Applied Biosystems) at 37°C for 1.5 h, rinsed in 70% formamide at 32°C for 10 min, and dehydrated in another ethanol series before re-probing at 37°C for 2 h. Following the second hybridization, slides were rinsed with 70% formamide at 32°C for 15 min followed by 5 min rinse in PN buffer. Chromosomes were counterstained with DAPI (4,6-diamidine-2-phenylindole dihydrochloride; Vectashield, Vector Laboratories). Preparations were examined and images captured and analyzed using a Zeiss Axioskop2 Plus microscope equipped with a Photometrics Coolsnap ES2 camera and running Metavue 7.1 software.