Immunofluorescent Microscopy for DNA Damage Detection

Immunofluorescent microscopy was conducted according to previously reported procedures with modifications [5 (link)]. Briefly, 2 × 10⁴ cells were seeded into 35 mm dishes containing a glass cover slip in each well. After irradiation, slides were air-dried, and fixed for 0.5 h in 2% paraformaldehyde in TBS. Cells were rinsed in TBS, placed in −20°C methanol for 1 min, rinsed, then placed for 20 min in TBS plus 1% bovine serum albumin and 0.2% Tween-20 (TTN) and finally incubated for 2 h with diluted antiphosphohistone H2AX (Ser-139) mAb (Upstate, Lake Placid, NY) diluted 1:500 in TTN. Slides were washed and incubated with FITC-conjugated anti-mouse goat F (ab’) ² fragment (DAKO, Carpinteria, CA) diluted 1:200 in TTN for 1 h at room temperature. Slides were rinsed and then immersed in 0.05 mg/ml DAPI for 15 min, rinsed and mounted with cover slips using 10 μl Fluorogard (Bio-Rad) as the antifade mounting medium, and sealed. To prevent bias in selection of cells that display foci, over 800 randomly selected cells were counted. Cells with three or more foci of any size were classified as positive. All experiments were repeated in triplicate.