Mouse anti γh2ax antibody

For the analysis of formation and repair of DNA double-strand breaks during and after irradiation, 50,000 MMTV-PyMT WT or K_Ca3.1 KO cells were seeded for 72 h in twelve-well chambers (ibidi, Planegg, Germany) in triplicate. TRAM-34 or its solvent was applied 1 h prior to irradiation with a single fraction of 0, 2, 4, or 6 Gy. After further 30 min or 24 h of culture, cells were fixed with 70% ethanol at −20°C for 10 min. Then, the cells were washed twice with 250 µL PBS and unspecific epitopes were blocked with 10% normal goat serum (Linaris, Dossenheim, Germany) in PBS for 1 h. Blocking solution was removed and the cells were incubated for 2 h in a primary mouse anti-γH2AX antibody (613402, BioLegend, London, UK) solution (1:500 dilution in 1.5% normal goat serum/PBS). Next, cells were washed twice with PBS and incubated with a secondary Alexa Fluor^® 555 goat anti-mouse antibody (A21127, Thermo Fisher Scientific) diluted 1:800 in 1.5% normal goat serum in PBS for 1 h. After two final PBS washing steps, the silicone chamber walls were removed, and the slide was cover-slipped with DAPI-containing vectashield solution. Four micrographs of different areas were taken using the F-view II camera (SIS, Munster, Germany) attached to fluorescence microscope (Olympus, Hamburg, Germany) and cell nuclei as well as the number of γH2AX foci per cell were counted.

Cells were seeded at 5000 cells/well in 6-well chamber slides (Lab-Tek) and grown to 60–80% confluency. After INC280 treatment for 18 h, cells were washed with PBS, fixed with 4% paraformaldehyde, permeabilized in 0.5% Triton X-100 for 20 min, and blocked in 5% BSA for 1 h at room temperature. To perform immunofluorescence (IF) staining, cells were incubated with mouse anti-γH2AX antibody (1:125 in 5% BSA, BioLegend) and rabbit anti-53BP1 antibody [1:100 in 5% BSA, Cell Signaling Technology (CST)] overnight at 4 °C. After washing 3 times in PBS cells were incubated with goat anti-mouse/Alexa488 and anti-rabbit/Alexa568 antibody (1:500 in PBST, Invitrogen) for 2 h. The nuclei were stained with DAPI (1 µg/ml, Thermo). Images were captured using an EVOS fluorescence microscope (Thermal Fisher).