2.9. Immunofluorescence: phospho‐histone h3 (phh3) and phospho‐histone h2ax (γ‐h2ax) staining

In order to confirm the induction of mitotic arrest and/or DNA damage after Plk1 inhibition, immunofluorescence experiments using the mouse monoclonal anti‐pHH3 (Ser10) antibody (1 : 2000, Merck Millipore, no. 05‐806) and anti‐γ‐H2AX (Ser139) antibody (1 : 500, Merck Millipore, no. 05‐636‐AF488) were performed. Seventy‐two hours (γ‐H2AX) or 24 h (pHH3) after treatment with volasertib (0–50 nm), cells were fixed with ice‐cold methanol, permeabilized with 0.1% Triton X‐100/PBS, and blocked with 1% BSA/PBS for 1 h. Next, cells were incubated overnight with the primary antibody at 4 °C, followed by 1‐h incubation at room temperature with the secondary antibody, that is, donkey anti‐mouse IgG Alexa Fluor^® 555 conjugate (1 : 1000, Thermo Scientific). Slides were counterstained with DAPI and mounted. Images of sections stained with the anti‐pHH3 antibody were taken using an Olympus BX51 standard research fluorescence microscope (Olympus, Aartselaar, Belgium), equipped with an Olympus DP71 digital camera (Olympus). Sections stained with the anti‐γ‐H2AX antibody were visualized with an Evos Cell Imaging System (Thermo Scientific). The percentage of positive pHH3 cells and the amount of γ‐H2AX foci per cell were counted using imagej software.