Ab2839

CC cells were seeded in 8-well chambers (Ibidi, Germany) at a density of 3000 /well. And CC cells were treated with 6 μg/ml CDDP for 1 h for DNA double-strand break staining (γH2AX fluorescence). Target cells were then fixed with 4% polyformaldehyde for 30 min, permeabilized with 0.1% TritonX-100 for 10 min and blocked with 10% BSA for 1 h at room temperature. Blocked cells were incubated overnight with primary antibodies against CEBPD (1:25; sc365546; Santa Cruz), IPO4 (1:50; ab181037; Abcam), γH2AX (1:50; ab2839; Abcam) at 4 °C and then labeled with Alexa Fluor-594-conjugated secondary antibody (1:200) for 1 h at room temperature. While the nuclei were stained for 2 min with DAPI (Sigma, USA). Confocal microscopy (LSM 510, META Laser scanning microscope, Zeiss) was used to acquire images. γH2AX fluorescence was quantitated using ImageJ (NIH, Bethesda, MD).

Cells were washed twice with PBS and fixed in 4% paraformaldehyde. The cells were incubated with rabbit polyclonal Ab against γH2AX (ab2839, Abcam) for 3 h at 37 °C, washed with PBS and incubated with DyLight 488-conjugated secondary antibody (Cwbiotech) for another 3 h. The cells were stained with DAPI (50 μg/ml) for 5 min for the detection of nuclei by Confocal FV1000 (Olympus). γH2AX-positive cell was calculated with (γH2AX add-in cells/DAPI stained cells) × 100%. At least 500 cells were counted per well.