Ab172580

Cells were washed with PBS and fixed with 4% formaldehyde at RT for 15 min. After fixation, cells were washed with 3% BSA/PBS and permeabilized with 0.5% Triton X-100/PBS at RT for 20 min. Next, cells were washed with 3% BSA/PBS and blocked in blocking buffer (10% FBS and 1% BSA in PBS) at RT for 1 h. Then cells were incubated with γH2AX (JBW301; Millipore; 1:500) and 53BP1 (ab172580; Abcam; 1:500) antibodies in the blocking buffer at 4 °C for overnight and washed with PBST. Finally, cells were incubated with goat anti-mouse IgG Alexa Fluor 488 and goat anti-rabbit IgG Alexa Fluor 568 (Thermo Fisher Scientific; 1:1000) in the blocking buffer at RT for 1 h, washed with PBST, and mounted in Vectashield containing DAPI (Vector Laboratories). Images were acquired using a Leica SP5 confocal microscope.

Intestinal paraffin samples were cut into 4 μm sections, deparaffinized, and stained with primary antibodies for cleaved caspase-3 (#9661; Cell Signaling Technology), cleaved PARP (#94885; Cell Signaling Technology), LC3B (#3868; Cell Signaling Technology), p62 (ab56416; Abcam), 4-HNE (ab46545; Abcam), HO-1 (ab137749; Abcam), Trx1 (#2429; Cell Signaling Technology), Gpx4 (MABF1969; Millipore), γH2AX (#9718; Cell Signaling Technology), 53BP1 (ab172580; Abcam), p-NF-κB p65 (ab86299; Abcam), collagen I (sc-59772; Santa Cruz), collagen III (ab7778; Abcam), occludin (GTX85016; Genetex), ZO-1 (GTX108592; Genetex), and claudin-2 (ab53032; Abcam). Images of the stained sections were acquired using the Olympus BX51 microscope with an Olympus DP72 camera and cellSens Standard 1.14 software (Olympus, Germany).