γ h2ax

Twenty-four hours following transfection with miR-20a-5p mimic or miRNA mimic negative control, 1 × 10⁵ cells were seeded in chamber slides and incubated overnight. The cells were subsequently exposed to 4 Gy irradiation (IR). Twenty-four hours following IR, the cells were fixed in 4% paraformaldehyde, permeabilized in 0.1% Triton X-100 (Sigma), blocked in 2% bovine serum albumin and incubated with a primary antibody against γ-H2AX (SanYing, China) overnight at 4 °C. The primary antibody was subsequently washed off, and a secondary antibody conjugated to fluorescein isothiocyanate was applied to the slides. Cells were washed with phosphate-buffered saline and counterstained with DAPI. The γ-H2AX foci were observed under a fluorescence microscope (Olympus). For each group, the γ-H2AX foci were counted in ≥50 cells.

DSBs represent an important ionizing radiation-induced lesion. The rapid phosphorylation of histone H2AX at serine 139 is a sensitive marker for DNA DSBs induced by ionizing radiation, which can later be detected by immunofluorescence.
The treated TE-1 cells were placed in 24−well plates with circular slides were fixed in 4% paraformaldehyde, permeabilized with 1% Triton X-100 for 10 min at room temperature, and then blocked with dilute 1% BSA (Solarbio, Beijing, China) for 1 h. The cells were incubated at 4°C overnight with the primary antibody γ-H2AX (Epitomics, Burlingame, CA, USA), which was diluted 1:1000 with 1% BSA. PBS was used to wash away the primary antibody. The cells were incubated with the secondary antibody at 37°C for 1 h, following which they were placed on coverslips and counterstained with 4-6-diamidino-2-phenylindole (Invitrogen) to mark the nuclei. Immunofluorescence staining was examined using a confocal microscope (Olympus Corporation) and then the mean number of γ-H2AX foci per cell (foci/cell) were counted.