Axioobserver z1 fluorescence microscope with apotome

Cells were fixed with fix/permeabilization solution (4% PFA and 0.2% Triton X-100 in PBS) for 15 min at room temperature after treatment and then blocked by block solution (2% normal goat serum (NGS) in PBS) for 30 min at room temperature. For γH2AX staining, cells were stained by γH2AX antibody (Alexa Fluor® 647 Mouse anti-H2AX (pS139), BD Bioscinces, USA, #AB_1645414), which coupled to Alexa Flour 647, at 1:50 dilution in block solution for 1 h at room temperature. For Rad51 staining, 1:100 Rad51 rabbit (Anti-Rad51 (Ab-1) Rabbit pAb, Millipore, USA, #PC130) in blocking solution was utilized to incubate for 30 min on a shaker at 4 °C. After that, anti-rabbit secondary antibody (Goat anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488, Thermo Fisher, USA, # A32731) with the dilution of 1:400 in blocking solution was used for staining for 75 min at room temperature. Nuclear foci were evaluated manually under the AxioObserver.Z1 Fluorescence Microscope (with Apotome) (Zeiss, Oberkochen, Germany).

Cells were fixed with fixation/permeabilization solution (4% PFA and 0.2% Triton X-100 in PBS) for 15 min at room temperature after treatment and then blocked with blocking solution (2% normal goat serum (NGS) in PBS) for 30 min at room temperature. For γH2AX staining, cells were stained with γH2AX antibody (Alexa Fluor® 647 mouse anti-H2AX (pS139), BD Biosciences, USA, #AB_1645414) coupled to Alexa Fluor 647 at 1:50 dilution in blocking solution for 1 h at room temperature. For Rad51 staining, 1:100 rabbit Rad51 (Anti-Rad51 (Ab-1) Rabbit pAb, Millipore, USA, #PC130) was used in blocking solution and incubated for 30 min on a shaker at 4 °C. DNA was stained with Hoechst33342 (3 µM in PBS) for 30 min at RT. Coverslips were mounted on glass slides with DAKO mounting medium (Dako NA Inc., Carpinteria, CA, USA). Nuclear foci were manually scored using the AxioObserver.Z1 fluorescence microscope (with Apotome) (Zeiss, Oberkochen, Germany). γH2AX foci in at least 50 cells per slide were counted.