Alexafluor 568 goat red anti rabbit igg

To detect mucin 2 (MUC2), Carnoy-fixed samples were cut into 5-µm-thick sections, mounted on adhesive microscope slides (SuperFrost Ultra Plus, Thermo Scientific), and rehydrated and rinsed in accordance with standard protocols^{17 (link),77 (link)}. The samples were confined (Dako Pen, Agilent Technologies) and incubated sequentially with a protein block (Dako, Agilent Technologies), a primary antibody (2 μg/mL of MUC2 rabbit polyclonal IgG, Santa Cruz Biotechnologies), and a secondary antibody (2 ng/mL of Alexafluor 568 goat red anti-rabbit IgG, Invitrogen, Thermo Fischer Scientific); both of the antibodies were diluted (Dako Diluent, Agilent Technologies). Sections were then treated with trihydrochloride trihydrate (0.5 mg/mL Hoechst 33342, Invitrogen, Thermo Fischer 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.) and Pannoramic Viewer and CaseViewer software (3DHISTECH Ltd.)

According to standard protocols, samples were fixed in 4% paraformaldehyde or Carnoy buffer, dehydrated, and embedded in paraffin^{4 (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.).

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.)