Gastric tissues sections were incubated at room temperature with 3 % hydrogen peroxide in PBS to block endogenous peroxidase and blocked for 1 h in a solution of 5 % human/mouse serum and 5 % BSA in PBS. Slides were then incubated with an anti-phosphoserine 139 of H2A histone family member X (pH2AX) polyclonal antibody (1:200; Novus) overnight at 4 °C followed by 30 min at room temperature with the EnVision+, HRP (Dako). Visualization was performed using 3,3′-diaminobenzidine, and tissues were counterstained by hematoxylin. The number of positive cells were determined in a blinded manner by our GI pathologist (M.B.P.).
AGS cells were fixed in 3.7 % paraformaldehyde, washed with PBS, and blocked with Universal Protein Block (Dako) for 40 min at room temperature. Cells were then incubated with the anti- pH2AX polyclonal antibody (1:200; Novus) overnight at 4 °C followed by 1 h at room temperature with the Donkey anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (1:600; Invitrogen). Slides were mounted with VECTASHIELD HardSet™ Antifade Mounting Medium with DAPI (Fisher Scientific) and confocal images were acquired using the Cytation C10 Confocal Imaging Reader (BioTek).
AGS cells were fixed in 3.7 % paraformaldehyde, washed with PBS, and blocked with Universal Protein Block (Dako) for 40 min at room temperature. Cells were then incubated with the anti- pH2AX polyclonal antibody (1:200; Novus) overnight at 4 °C followed by 1 h at room temperature with the Donkey anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (1:600; Invitrogen). Slides were mounted with VECTASHIELD HardSet™ Antifade Mounting Medium with DAPI (Fisher Scientific) and confocal images were acquired using the Cytation C10 Confocal Imaging Reader (BioTek).