Flushed colons were fixed in Carnoy buffer, dehydrated and embedded in paraffin following a standard protocol. Tissue blocks were cut into thin sections (5 µm) on a Leica RM2265 microtome and mounted on adhesive microscope slides (Superfrost ultra plus, ThermoScientific, Waltham, MA, USA). Histological features were analysed by Alcian blue (AB) staining [56 (link),57 (link)]. For mucin-2 (Muc2) detection, samples were isolated (Dako Pen, Agilent Technologies, Santa Clara, CA, USA) and incubated sequentially with: the Protein block (Dako, Agilent Technologies), the primary antibody (2 μg/mL, Mucin 2 rabbit polyclonal IgG, Santa Cruz Biotechnologies, Dallas, TX, USA) and the secondary antibody (2 ng/mL, Alexafluor 568 goat red anti-rabbit IgG, Invitrogen, ThermoFischer Scientific, Waltham, MA, USA) both diluted in Ab diluent (Dako, Agilent Technologies). Sections were then treated with trihydro-chloride trihydrate (0.5 mg/mL Hoechst 33342, Invitrogen, ThermoFischer Scientific) in PBS. The slides were mounted using fluorescent mounting medium (Dako, Agilent Technologies). Tissues were visualized using a high capacity digital slide scanner (3DHISTECH Ltd., Budapest, Hungary) and the Panoramic and Case viewer software (3DHISTECH Ltd.)
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