5-Bromo-2′-deoxy-uridine (BrdU) incorporation assays

HUVEC cells were subjected to hypoxic condition and chemical treatment for 6 h and then incubated with 0.1 mg/ml BrdU for 3 h before fixed by 4% paraformaldehyde (PFA). Cells were incubated in 0.5% Triton X-100, 1.5 N HCl, and washed by PBS between incubation, then add 0.25% trypsin EDTA, and incubate at 37 °C for 5 min. Cells were washed by PBS and blocked in blocking buffer for 2 h. BrdU antibody (1:100, ab6326, Abcam) was used and then incubated overnight at 4 °C in a hydration chamber. After wash with PBST, secondary antibody containing 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) were added and incubated at RT for another 3 h, then observed with Olympus IX81.