Vectastain elite abc kit for rabbit

The sequences, preparation and use of the small interference RNAs (siRNAs) used in this work have been already described [16 (link), 43 (link)]. The commercial synthetic siRNAs (siCD40 sense 5′-GUGUGUUACGUGCAGUGACUU, siCD40 antisense 5′-AAGUCACUGCACGUAACACACUG, siSC sense 5′-ACUACAAGACUCGUGACCAUU, siSC antisense 5′-UGGUCACGAGUCUUGUAGUUU, all from Mycrosynth, Switzerland) included stabilizing phosphorothioate backbone and 2′-O-methyl sugar modifications, as well as a cholesterol molecule added at the 3′ end of the sense strand with a pyrolidine linker to facilitate uptake and internalization.
For the immunochemical work, we used a rat monoclonal antibody against F4/80 (Hycult biotech, Uden, NL), a rabbit polyclonal anti-NF-κB-p65 (anti-phospho-S536, Ab86299, Abcam, Cambridge, UK), and a goat polyclonal anti-CD31/PECAM-1 (platelet-endothelial cell adhesion molecule-1) antibody (M-20, sc-1506, Santa Cruz Biotechnology, Santa Cruz, CA). As a secondary antibody we used a goat anti-rat IgG2 (Novus Biologicals, Littleton, CO), a biotinylated horse anti-goat IgG (BA-9500, Vector, Burlingame, CA) or a Vectastain Elite ABC kit for rabbit antibodies (PK-4001-NB, Vector Laboratory, Burlingame, CA).

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)].