temperature with 1 μg ml−1 Hoechst 33258 pentahydrate (H3569, Molecular Probes) in 2× SSC. The slides were then transferred to a Petri dish with 2× SSC and exposed to UV irradiation for 30 min in a Stratalinker Crosslinker (Stratagene). The slides were then dehydrated by passing them through an ethanol series (70%, 96% and 100%) and placed in PBS for 5 min at room temperature. The DNA was denatured by exposure to 0.07 N NaOH for 2 min at room temperature. The slides were then washed three times in PBS for 5 min. The slides were then blocked in PBS, 1% BSA, 0.5% Tween-20 for 1 h at room temperature and stained overnight with a FITC-conjugated mouse anti-BrdU antibody (Clone B44, BD Biosciences) diluted 1:1 in PBS, 3% BSA, 0.5% Tween-20 at room temperature. The slides were then washed three times with PBS, 1% BSA, 0.5% Tween-20 for 5 min at room temperature and stained with goat anti-mouse Alexa Fluor-488 secondary antibody (A-11001, Life Technologies) diluted 1:500 in PBS, 1% BSA, 0.5% Tween-20 for 6 h at room temperature. The slides were then washed three times in PBS, 1% BSA, 0.5% Tween-20 for 15 min and stained with Hoechst 33342
