8 oxog

For immunofluorescence staining, cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) for 10 min at room temperature and permeabilized with 0.5% Triton^™ X-100 (Bio Basic). Specific antibodies against 8-OxoG (Abcam), phospho-histone H2AX (Ser139; Millipore), phospho-CHK1 (Ser345), phospho-P53 (Ser15), PARP1, cleaved-caspase 3 (Cell Signaling Technology), and poly-ADP-ribose (Enzo Life Sciences) were used for visualization of the proteins. The images were captured using a fluorescence microscope (Nikon) equipped with the NIS-Elements 4.0 Nikon imaging software. For quantification, a minimum of 500 cells were analyzed from each of three independent experiments.

Immunostaining was performed according to prior reports^{[1 (link)]}. Briefly, heart cryosections were equilibrated with antigen retrieval buffer in epitope retrieval buffer (IHC World) or 1× citrate buffer (Antigen Retrieval Citra Plus, Biogenex). Samples were permeabilized and blocked with 0.3% Triton X-100 and 10% serum from the host animal of secondary antibodies in PBS for 1 h at room temperature. Then, the samples were incubated overnight at 4 0C with primary antibodies. After three washes in PBS, samples were incubated at room temperature for 1h with the corresponding fluorescence secondary antibodies conjugated to Alexa Fluor 488 or 555 (Invitrogen) at 1:400. The slides were mounted in Vectashield Antifade Mounting Medium (Vector Laboratories). Slides were viewed under Nikon fluorescence or Zeiss LSM 510 confocal microscopes. Primary antibodies: pH3 Ser10 (EMD Millipore, 06–570; 1:100); troponin T (Thermo Scientific, MS-295-P1; 1:200); 8-oxoG (Abcam ab64548, 1:25). DAPI was used for nuclear staining. Images were obtained on a Nikon Eclipse Ni or Nikon A1 laser scanning confocal microscopes.