2.4. Immunofluorescence—γ-H2AX Assay

In brief, cells were washed in phosphate buffered saline (PBS) then fixed using 4% paraformaldehyde (PFA) in PBS for 15 min. In the next step, cells were permeabilized using 0.2% Triton-X solution (Sigma-Aldrich) in dH₂O for 10 min at 4 °C. Then, non-specific sites on the cells were blocked using 100 μL 2.5% bovine serum albumin (BSA) blocking buffer for each slide. Slides were covered with parafilm and placed in a humidified dark box for one hour. Next, a total of 100 μL of diluted γ-H2AX antibody conjugated with Alexa Fluor (Merck Millipore, Burlington, MA, USA), at the relevant concentration (1:100) (following manufacturer’s instructions), was added to the slides. The slides were recovered with parafilm and placed in a humidified dark box for one hour. After that, the slides were washed 3 times for 5 min in TBST (Tris-buffered saline with Tween 20, PH7.5, Sigma-Aldrich) and then PBS. The slides were de-hydrated in gradient ethanol series for 5 min each time. Once the slides were air dry, 15 μL DAPI (Vector Laboratories, Burlingame, CA, USA) was added and each slide covered with a cover slip and sealed using clear nail varnish. The slides were analysed under a DM4000 microscope (Leica, Milton Keynes, UK).