(PFA EMS #15710) in cold phosphate-buffered saline (PBS) and permeabilized with
0.5% Triton X-100 with 3% bovine serum albumin (BSA) for 20 min at room
temperature. The sections or cells were incubated with primary antibodies
overnight at 4°C (γ-H2A.X clone JBw1 Merk05-636) and with Alexa-conjugated
secondary antibodies (1/300) and Hoechst 33342 for 45 min. Sections were then
analyzed using an automated Axioscan (Zeiss) or inverted Observer.Z1 Apotome
microscope (Zeiss). For 5-bromo-2′-deoxyuridine (BrdU) immunostaining, cells
were fixed with 4% PFA, washed and unmasked with 2 N HCl for 20 min at room
temperature, neutralized with 0.1 M borate, and then processed as described for
the other primary antibodies.