Bond max slide stainer

Pancreatic tissues were fixed overnight in 10% neutral buffered formalin, paraffin-embedded, and sectioned at 5 μm. The sections were stained with hematoxylin and eosin. For immunohistochemistry, after dewaxing, rehydrating tissue paraffin sections, and antigen retrieval by pretreatment with high temperature at pH 9, the sections were immunolabelled using an automated immunohistochemical stainer according to the manufacturer’s guidelines (Bond-Max slide stainer, Menarini, Leica Microsystems). For OX₁R assessment, 3μm pancreatic tissue sections were incubated for 30 minutes with a polyclonal anti-OX₁R antibody (Life technologies, Saint Aubin, France; polyclonal rabbit 1/100, PAS-33837), rinsed, and then incubated with a biotinylated secondary donkey anti-rabbit antibody diluted at 1:200. Sections were rinsed and incubated with Streptavidin (TrekAvidin-HRP; Biocare Medical) and diaminobenzidine ultraview detection kit (Bond Polymer Refine detection; DS9800; Leica Microsystems). Substitution of the primary antibody with PBS was used as a negative control.

Skin samples were fixed in 4% neutral buffered paraformaldehyde solution at room temperature for at least 24 h, embedded in paraffin blocks (Shandon Cytoblock, Thermo Scientific, USA), cut into 4 μm sections and stained with haematoxylin, eosin and saffron (H&E). Immunohistochemical procedures were carried out using an automated immunohistochemical apparatus according to the manufacturer’s instructions (Bond-Max slide stainer, Leica Microsystems).
Briefly, after dewaxing, paraffin sections were rehydrated, and antigen retrieval was performed in an Antigen Retrieval Buffer (Leica Biosystems) at pH 9. Sections were incubated for 30 min with primary antibodies, rinsed, and then incubated with a biotinylated secondary antibody. Sections were rinsed and the reaction was developed according to the manufacturer’s guidelines (streptavidin-peroxidase with an automated BOND, Leica Microsystems). Primary antibodies were used at the indicated dilutions: GATA6 (1:750, D61E4 clone, Cell Signalling 5851), Ki67 (1:100, Clone MIB-1, Dako M7240) and KRT5/6 (1:100, Clone D5/16 B4, Dako M7237). Biotinylated secondary antibodies (Vector Laboratories) were used at 1:400 dilution. Images were acquired with a Pannoramic 250 Flash slide scanner (3DHistech) and visualised with QuPath version 0.1.2 (https://qupath.github.io).