Mouse anti brdu antibody

At 68 h after doxycycline inducement, zebrafish larvae were incubated in 10 mM BrdU (Sigma, St. Louis, MO, USA) in 1% DMSO for four hours at 28.5 °C, then anesthetized in tricaine and fixed with 4% paraformaldehyde for two hours at RT. The fixed embryos were then sent for cryosectioning. Sections were treated with 2 N HCl for 1 h at RT, blocked 1 h with blocking solution, then incubated with mouse anti-BrdU antibody (Invitrogen) overnight at 4 °C. The next day, after a 1-h wash with PBST, the sections were further incubated with secondary goat anti-mouse IgG antibody. For cell culture analysis, Hep3B cells were incubated with BrdU at a final concentration of 20 μM for 2 h. After a PBS wash, cells were fixed with 4% PFA for 5 min, permeabilized with acetone:methanol (1:1) for 3 min on ice, then rehydrated in PBS for 5 min. The following denaturing and antibody incubation steps were similar to those of the staining of the cryosections.

WT and MDPL-HDFs were cultured and treated with cisplatin for 24 hours. After 24 and 48 hours from the end of treatment, cells were incubated for 6 hours with 10 μM BrdU (Life Technologies Corporation). Then they were collected, fixed with cold ethanol 90%, added with 1 mL of 2 N HCl + 0.5% Triton X-100 and resuspended with 1ml of 0,1 M borate buffer. Finally, pelleted cells were suspended with 200 μl of washing buffer + 1:50 mouse anti-BrdU antibody (Invitrogen, Life Technologies Corporation, CAT#33900, RRID: AB_86146) and incubated for 45 min at room temperature followed by two washes. Subsequently cells were incubated with FITC-conjugated secondary antibody (1:800) for 30 min at +4° C. For DNA content assay, cells were successively stained with propidium iodide (Sigma-Aldrich, St. Louis, MO, USA). Cytofluorimetric acquisition was done on a on a Gallios instrument using Kaluza G Acquisition software and the analyses were performed using Kaluza Analysis Software v.2.1 (all from Beckman Coulter, Inc.).