4 hydroxynonenal antibody

ApoE KO and Nox4ApoE DKO mice were received intravenous injection of saline or rFliC (25 μg/mouse). After 2 hrs, the thoracic aorta were isolated and were embedded in Frozen Section Compound (Leica), frozen on dry ice. Five cryosections (each 10 μm thick) were obtained in each animal. The samples were incubated with 4-hydroxynonenal antibody (Abcam) at 4 °C for overnight. An Alexa Fluor® 594-conjugated goat anti-mouse IgG was used as the second antibody.

Stomach tissue collection was performed as described previously (10 (link)). Briefly, the stomachs were opened along the greater curvature. For histology, gastric strips from both the lesser and greater curvatures were fixed in formalin for paraffin sections. For DNA, gastric tissue was snap frozen in liquid nitrogen and extracted using the DNEasy Blood and Tissue Kit (Qiagen, Valencia, CA). For flow cytometry, gastric cells were digested using a modified version of the protocol described by Geem et al. (11 (link)), which utilizes 17.9 μg/ml Liberase TM (Cat #05401119001, Roche Diagnostics Corporation, Indianapolis, IN) instead of Type VIII collagenase. Immunohistochemistry and immunofluorescence were performed as described previously (10 (link)), using antibodies against the following targets: calprotectin (#ab22506, Abcam); CD11b-AF594 (clone M1/70, #101254, BioLegend); Cxcr2-AF488 (#FAB2164G, R&D Systems); GSII-FITC lectin (#FL-1211, Vector Labs, Burlingame); H⁺/K⁺-ATPase-β (#D032-3, Medical and Biological Laboratories, Woburn, MA); intrinsic factor (gift from David Alpers, Washington University, St. Louis, MO); E-cadherin-FITC (#612130, BD Biosciences, San Jose, CA); E-cadherin-AF647 (#560062, BD Biosciences); and 4 Hydroxynonenal antibody (#ab46545, Abcam).