Rabbit anti h pylori antibody

The oral vaccines were prepared by using 100 μg of FVpE in 500 μl polysaccharide adjuvant (PA) containing LBP (20 μg/mL) and chitosan (1%, w/w). Closed ileal loop and immunohistochemical analysis were performed to verify whether PA could facilitate the delivery of FVpE to Peyer's patches (24 (link)). Briefly, 6 week-old male SPF BALB/c mice were fasted overnight. Then, the mice were sacrificed and the closed ileal loops containing one or two Peyer's patches were prepared. An equivalent amount of FVpE protein with or without PA, was injected into the lumen of ileal loops. After incubation for 1 h, the ileal loops were excised and washed with PBS. The tissue samples were then fixed and freeze-sectioned. The FVpE protein were detected using rabbit anti-H. pylori antibody (Abcam, UK) and Goat anti-rabbit IgG conjugated with Alexa Fluor® 647 (Abcam, UK). A well-known M cell specific antibody, anti-Gp2 monoclonal antibody conjugated with Alexa Fluor® 488 (MBL, Japan), was used for localization of M cells in Peyer's patch. Besides, nuclei were stained by DAPI (Sigma, USA).

H. pylori were detected in infected gastric tissues by immunohistochemistry (IHC) using a rabbit anti-H. pylori antibody (Abcam, Milton, Cambridge, UK). Positive-staining cells were visualized with diaminobenzidine (DAB) (Vectastain Elite ABC Kit for rabbit; Vector Laboratories, Burlingame, CA, USA) and morphometrically analyzed with Leica DM 2500 microscopy and Leica Application Suite software (version 4.4; Leica microsystems, Heerbrugg, Switzerland). The evaluation of H. pylori-positive cells as marker of the infection was performed by counting the H. pylori-positive cells distributed in the non-infected control gastric tissue. Paraffin embedded longitudinal sections of antrum and corpus were stained with hematoxylin and eosin (H&E) and evaluated. It was graded for gastritis and mucosal changes and analyzed by a double blind test according to the grading scheme for rodents [32 (link),37 (link)].

Cells treated with compounds, enzymes, OMVs, blocking antibodies, or inhibitors were infected with H. pylori 26695 (MOI = 50) at 37 °C for 1 h. After the cells were washed to remove non-adherent bacteria, they were detached with 2 mM EDTA and fixed with 2% formaldehyde. Cells with plasma membrane-associated H. pylori bacteria were stained with rabbit anti-H. pylori antibody (Abcam, 1:1,000) at 4 °C for 16 h and then washed with PBS. The secondary antibody was fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit antibody (Invitrogen, 1:1000), which was applied at 4 °C for 1 h, and the samples were then washed with PBS. Cells were analyzed by a flow cytometry system (BD FACSCalibur Calibur Flow Cytometer 4 Color). BD FACStation Software (Version 3.3) was used for the data acquisition. Adherence was quantitated in terms of the proportion of cells with adherent H. pylori. At least over 10,000 cells were collected. For gating strategy, as shown in Supplementary Fig. 5, cell debris was first removed by gating the main cell population using the FSC/SSC gating. Mock-treated cells (cells only without bacterial infection) that had been treated with the same staining procedure as aforementioned were used to distinguish the boundary between the positive or negative staining populations. An identical threshold was applied for all samples within the same cell line.