Rbk012

For the immunohistochemical study, sections were deparaffinised, hydrated and antigen retrieval was performed in a pressure cooker in 10 mmol/L sodium citrate buffer, pH 6.0, for 2 minutes (min). Slides were cooled for 10 min at room temperature and rinsed twice in triphosphate buffered saline (TBS) for 5 min. The NovolinkTM Max-Polymer detection system (Novocastra, Newcastle, UK) was used for visualisation, according to the manufacturer’s instructions. After blocking endogenous peroxidase with 3% hydrogen peroxide in methanol for 10 min, sections were incubated overnight at 4°C, with a polyclonal antiserum against H. pylori (RBK012; Zytomed, German) which shows immunoreactivity with a wide range of bacteria belonging to the Helicobacter genus. Sections were rinsed with TBS between each step of the procedure. Colour was developed for up to 7 min at room temperature with 3,3′-diamino-benzidine (DAB) (Sigma, St. Louis, MO) and sections were then lightly counterstained with haematoxylin, dehydrated and mounted. Positive immunoreactivity was recorded as a distinct golden-brown labelling of the bacteria located on mucosal surface, in gastric pits or glands and in parietal cells.

Sections were deparaffinized in xylene and rehydrated in sequential graded alcohols, and antigen retrieval was accomplished in a water bath in a 10 mmol/L sodium citrate buffer at pH 6.0 for 20 minutes (min). Slides were chilled for 10 min at room temperature and washed twice for 5 min in triphosphate buffered saline (TBS). For visualization, the NovolinkTM Max-Polymer detection system (Novocastra^®, Newcastle, UK) was used, following the manufacturer’s instructions. Sections were incubated overnight at 4 °C, with a polyclonal anti-serum against H. pylori (RBK012; Zytomed, Berlin, German) (1:200) that has been shown to have immunoreactivity with a wide assortment of Helicobacter genus bacteria [4 (link)]. Sections were rinsed with TBS between each step of the procedure. Color was developed with 3,3′-diamino-benzidine (DAB) (Sigma^®, St. Louis, MO, USA), and sections were then lightly counterstained with hematoxylin, dehydrated, and mounted. Negative controls were performed by replacing the primary antibody with an antibody of the same immunoglobulin isotype at the same original concentration. Positive immunoreactivity was recorded as a distinct golden-brown labelling of the bacteria located on the mucosal surface, in gastric pits or glands, and inside parietal cells.