Quantifying H. pylori-Induced c-Jun Activation

Monolayers of primary gastric epithelial cells derived from C57BL/6^WT, C57BL/6^Nod1−/−, FVB/N INS-GAS^Nod1+/+, and FVB/N INS-GAS^Nod1−/− mice were infected for 1 hour with H. pylori strains 7.13 or PMSS1. After infection, monolayers were subjected to c-Jun immunofluorescence staining as previously described (26 (link)). Briefly, cells were fixed with 4% paraformaldehyde (Thermo Scientific) for 1 hour and then blocked with 5% goat serum (Sigma) in PBS for 1 hour. Samples were incubated with an anti-phospho-c-Jun antibody (1:500 dilution; Cell Signaling) overnight at 4°C. Samples were then incubated with Alexa 488-anti-rabbit (1:1000; Invitrogen), Alexa 546-Phalloidin (1:500; Invitrogen), and Hoechst 33342 (1:1000; Invitrogen) for 1 hour at room temperature. Slides were mounted using ProLong Glass (Invitrogen), and images were acquired in an Olympus FV-1000 confocal microscopy.