Cells were seeded into 8-well μ-Slides (Ibidi) at a density of 80,000 cells/well. After overnight incubation, cells were washed with pre-warmed PBS, fixed with 4% formaldehyde solution for 15 min at room temperature, washed, and then blocked with complete blocking solution (0.5% BSA, 0.1% TritonX-100, 5% goat serum in sterile PBS) for one hour at room temperature. Next, samples were incubated overnight at 4 °C with the relevant primary antibodies (anti-E-Cadherin antibody (ab11512-Abcam), anti-Vimentin Antibody (V9) (sc-6260-SantaCruz), anti-gamma H2A.X (phospho S139) antibody (ab11174-Abcam) anti-cytokeratin 8 (ab53280-Abcam) and anti-cytokeratin 14 (ab7800-Abcam). After incubation, the cells were washed with PBS, and the secondary antibodies (Alexa Flour 488, Alexa Fluor 546, Alexa Fluor 555) were added in complete blocking solution, followed by incubation for two hours at room temperature. Nuclei were labeled with DAPI. Imaging was carried out using a ZEISS LSM-710 system (Carl Zeiss microscopy Gmbh, Jena, Germany) with a 40×/1.4 Plan-Apochromat oil immersion objective. Images were processed with ZEN (Carl Zeiss microscopy Gmbh, Jena, Germany). γ-H2AX foci were counted with FindFoci, an automated ImageJ plugin [96 (link)].
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