γh2ax antibody

Western blotting was performed using a quantitative Western blot system (LICOR Bioscience, Lincoln, NE, USA) as previously described [23 (link), 26 (link)]. The primary antibodies are listed in Supplementary Table 1. For immunofluorescence, A172 cells were grown on coverslips and GBM1A neurospheres were collected by cytospin onto glass slides. Cells were fixed with 4% paraformaldehyde and immunostained with γH2AX antibody (Cell Signaling) according to manufacturers’ protocols, followed by secondary antibody. Secondary antibodies were conjugated with Cy3. Coverslips were treated with Vectashield antifade solution containing 4′6-diamidino-2-phenylindole (Vector Laboratories). Tumor cell proliferation and apoptotic cells were assessed by Ki-67 and cleaved caspase-3 immunohistochemistry, respectively. Frozen tumor sections (7–8 μm thick) were immunostained with primary antibodies against Ki-67 and cleaved caspase-3 (Cell Signaling, Danvers, MA, USA) as previously described [22 (link), 27 (link), 28 (link)]. Apoptotic and cell proliferation indices were determined by computer-assisted quantification of the number of positively stained cells per microscopic field as previously described [22 (link), 29 ].

HFF-1 were seeded on top of glass cover slips for immunofluorescence experiments and fixed in 4% paraformaldehyde. The cells were permeabilized in phosphate buffered saline (PBS) containing 0.1% sodium citrate and 0.3% Triton-X and blocking was performed in 1% bovine serum albumin in PBS. The cells were incubated in γH2AX antibody (#2577S, Cell Signaling) overnight at 4 °C. Alexa Fluor anti-rabbit 488 was used as a secondary antibody. The nuclei were counterstained with 4′, 6-diamidino-2-phenylindole (DAPI) and the cells were analyzed using Cell Voyager CV1000 Yokogawa (Visitron Systems). Quantification of nuclear γH2AX fluorescence intensity was performed using ImageJ software.

Cells were plated in 35-mm glass bottomed culture dishes (Mattek), and were cultured overnight. The cells were then left untreated (DMSO) or treated with 10 μM Rucaparib or iRucaparib for 48 hrs before washing with PBS. Cells were fixed with 4% Paraformaldehyde for 15 min at RT and were washed three times with PBS. The cells were then permeabilized with 0.1% Triton X-100 in PBS for 5 min and were blocked with 1% BSA in PBS for 30 min. Fixed cells were incubated with a γH2AX antibody (1:200, Cell Signaling Technology, #9718) at 4℃ for overnight, washed three times with PBS for 5 min, and incubated with an Alexa Fluor 488 conjugated anti-rabbit antibody (1:500, Thermo Fisher Scientific, A-11008) for one hour at RT. Cells were washed three times with PBS for 5 min and stained with DAPI (1:1000, Thermo, #62248) for 2 min. Cells were washed with PBS and mounted with the FluorSave reagent (Millipore, #345789). Images were collected on a Zeiss LSM 880 Airyscan inverted confocal microscope.