Immunofluorescence Assay for Nuclear Markers

Immunofluorescence was performed as described previously^{5 (link)}. Briefly, cells were fixed in 4% paraformaldehyde in PBS for 30 min at room temperature. Cells were washed twice with PBS, and permeabilized with 0.5% Triton X-100 in PBS for 10 min. After washing two times, cells were blocked in 10% BSA in PBS for 1 h at room temperature. Cells were then incubated with primary antibodies in 5% BSA in PBS supplemented with 0.1% Tween 20 (PBST) overnight at 4 °C. The next day, the cells were washed four times with PBST, each for 10 min, followed by incubation with Alexa Fluor-conjugated secondary antibody (Life Technologies), in 5% BSA/PBST for 1 h at room temperature. The cells were then washed four times in PBST, incubated with 1 µg/ml DAPI in PBS for 5 min, and washed twice with PBS. The slides were mounted with ProLong Gold (Life Technologies), and imaged with Leica TCS SP8 fluorescent confocal microscope. Quantification of % positive cells of SAHF, p-ATM, IL8, γH2AX, and p65 was done under identical microscopy settings between samples. Cells with over 5 visible spots at the expected location were considered positive. Over 200 cells from 4 randomly-selected fields were analyzed.