According to standard protocols, samples were fixed in 4% paraformaldehyde or Carnoy buffer, dehydrated, and embedded in paraffin4 (link). Histological analyses were carried out on tissue sections blocks of 5 µm cut on a Leica RM2265 microtome and mounted on adhesive microscope slides (Superfros, ultra plus, ThermoScientific). Samples were then stained with hematoxylin–eosin Safran, periodic acid-Schiff (PAS), and Alcial blue (AB)27 (link),28 (link).
For mucin-2 (Muc2) detection, sections were deparaffinized, rehydrated, and rinsed according to standard protocols. Then, samples were incubated sequentially with the protein block (Dako, Agilent Technologies), the primary antibody (2 µl/mL, Mucin 2 rabbit polyclonal IgG, Santa Cruz Biotechnologies), and secondary antibody (2 ng/mL, Alexafluor 568 goat red anti-rabbit IgG, Invitrogen, ThermoFischer Scientific) both diluted in the Ab diluent (Dako, Agilent Technologies). The sections were then treated with trihydro-chloride trihydrate (0.5 mg/mL Hoechst 33342, Invitrogen, ThermoFischer Scientific) in PBS. Subsequently, the slides were mounted using Fluorescent Mounting Medium (Dako, Agilent Technologies).
Tissues were visualized using a high-capacity digital slide scanner (3DHISTECH Ltd.) and Panoramic and Case viewer software (3DHISTECH Ltd.).
For mucin-2 (Muc2) detection, sections were deparaffinized, rehydrated, and rinsed according to standard protocols. Then, samples were incubated sequentially with the protein block (Dako, Agilent Technologies), the primary antibody (2 µl/mL, Mucin 2 rabbit polyclonal IgG, Santa Cruz Biotechnologies), and secondary antibody (2 ng/mL, Alexafluor 568 goat red anti-rabbit IgG, Invitrogen, ThermoFischer Scientific) both diluted in the Ab diluent (Dako, Agilent Technologies). The sections were then treated with trihydro-chloride trihydrate (0.5 mg/mL Hoechst 33342, Invitrogen, ThermoFischer Scientific) in PBS. Subsequently, the slides were mounted using Fluorescent Mounting Medium (Dako, Agilent Technologies).
Tissues were visualized using a high-capacity digital slide scanner (3DHISTECH Ltd.) and Panoramic and Case viewer software (3DHISTECH Ltd.).
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