Hoechst dna stain

Cells were seeded on top of poly-L-lysine coated glass coverslips. After treatment, coverslips were rinsed 2× with PBS, crosslinked in 3.7% formaldehyde in PBS, washed 3× for 5 min in PBS/0.1% Triton-X. Cells were blocked with PBS/0.1% Triton-X/3% BSA for 30 min at RT. Cells were incubated with primary antibody against phospho-Histone H2A.X (1:100, Millipore Sigma) in blocking solution and washed 3× for 5 min in PBS. Secondary antibody (anti-mouse labeled with Alexa Fluor 488, 1:800 in blocking solution, ab150113, Abcam) was incubated for 1 h at RT. Cells were washed 3× for 5 min in PBS, 15 min in PBS with Hoechst DNA stain (1:1000; Thermo Fisher Scientific), rinsed with PBS and mounted with ProlongGlass (Life Technologies) on top of a glass slide.

We plated HeLa and 3T3 cells on poly-l-lysine-coated coverslips. Forty-eight hours post-transfection, we rinsed the coverslips twice with PBS and fixed the cells with 3.7% formaldehyde in PBS for 10 min at room temperature. After 2 washes with PBS, we permeabilized the cells with PBT (PBS, 0.1% Tween-20) for 15 min at room temperature. Next, we blocked the coverslips with 5% BSA in PBT for 1 h at room temperature and then incubated the coverslips with properly diluted primary antibody (mouse M2 monoclonal ANTI FLAG, 1:800, F1804, Sigma; rabbit polyclonal Tom20, 1:800, FL-145, Santa Cruz) in 5% BSA in PBT for 3 h at 37 °C in a humid chamber. Coverslips were washed three times for 5 min each with PBT and incubated with diluted secondary antibody (anti-mouse labeled with Alexa Fluor 488, 1:800, ab150113, Abcam; anti-rabbit labeled with Alexa Fluor 647, 1:800, 4414S, CST) in 5% BSA in PBT for 1 h at room temperature. Cells were then washed twice for 5 min with PBS, once for 20 min with PBS containing Hoechst DNA stain (1:1000, Thermo Fisher Scientific), rinsed in PBS, and then mounted on glass slides with ProLong Gold (Thermo Fisher Scientific).