Bromodeoxyuridine labeling

Cells were grown and labeled with a thymidine analog BrdU for 48 h together with treatments. The complete medium of monolayer was replaced with medium containing 40 μM 5-Bromo-2′-deoxyuridine (Sigma), 5 μM uridine (Sigma) and 0.4 μM 5-fluoro-2′-deoxyuridine (Sigma). After treatments, the cells were fixed with fixation solution (2% formaldehyde - 0,2% glutaraldehyde) for 10 min. Then, samples were digested by Hind lll on React 2 buffer (Gibco) and Eco Rl on SH buffer (Sigma) for 1 h to create single-stranded regions in the DNA and to expose the incorporation of BrdU to the monoclonal mouse antibody [36]. After this, the samples were finished as previously described in a conventional immunofluorescence assay. Results are expressed as an index of BrdU positive nuclei relative to total number of nuclei.