Quantification of Colonic Muc2 Protein

Mucin 2 (Muc2) proteins were labelled on formalin-fixed samples that were cut, using a microtome (Leica RM2255, Leica, Wetzlar, Germany), into 5-μm-thick sections and mounted on adhesive microscope slides (Adhesives slides KP printer, KliniPath, Duiven, The Netherlands). Immunostaining was performed using the Leica Bond RXm. Heat-induced antigen retrieval was done with Leica Bond™ Epitope Retrieval 1 (pH6) for 20 min. Rabbit polyclonal antibody against the MUC2 protein (NBP1-31231, Novus Biologicals, Littleton, CO, USA), diluted at 1/500, was revealed by a goat anti-rabbit Alexafluor 568 IgG (dilution 1/2000, Invitrogen, Waltham, MA, USA) with 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI, Invitrogen, Waltham, MA, USA) to counterstained nuclear. All the reagents were diluted with Bond Antibody Diluent (AR9352, Leica, Wetzlar, Germany). Slides were mounted using a fluorescent mounting medium (Fluoromount-G, Clinisciences, Nanterre, France), scanned with the Panoramic Scan 150 (3D Histech, Budapest, Hungary) and analyzed with the CaseCenter 2.9 viewer (3D Histech, Budapest, Hungary). Ten crypt (colon) or villus/crypt (ileum) units were analyzed in each sample, and the number of Muc2 cells was counted according to Fukuda et al. [17 (link)]. Tissue samples with non-well-oriented crypts were removed from the analysis.