C2 confocal laser scanning microscope system

Frozen duodenal biopsy samples derived from controls and children with CD were embedded into Shandon cryomatrix (ThermoElectron Co., Madison, WI, USA) and cut into 5 μm sections. Samples were incubated with primary antibodies specific for human IL-24 (ab182567; rabbit, 1:100, Abcam, Cambridge, United Kingdom) or IL-20RB (ab124332; rabbit, 1:100, Abcam) for 1 h at room temperature (RT). After repeated washing, slides were incubated with the corresponding Alexa Fluor 568 secondary antibody (1:200 anti-rabbit, Invitrogen) for 30 min at RT in the dark and counterstained with Hoechst 33342 (1:2000, Sigma-Aldrich). Finally, sections were coverslipped with Vectashield fluorescent mounting medium (Vector Laboratories, Burlingame, Calif., USA). Appropriate controls were performed by omitting the primary antibody to assure specificity and to avoid autofluorescence. Sections were analyzed with a Nikon C2 confocal laser scanning microscope system (Nikon, Minato, Tokyo, Japan).

HT-29 cells were seeded in chambers and cultured for 24 h at 37 °C. After repeated washing, cells were permeabilized with Cytofix/Cytoperm (BD Pharmingen, San Diego, CA, USA) for 15 min at room temperature (RT), and were washed again. Human frozen colon biopsy samples were embedded into Shandon cryomatrix (ThermoElectron Co., Madison, WI, USA) and cut into 5 μm sections. Thereafter cells or tissue sections were incubated with rabbit anti-human PARK7 or IL-17R polyclonal IgG primary antibody (Abcam, Cambridge, United Kingdom) (1:200) for 1 h at RT. Cells were washed with WashPerm solution, and tissue sections with PBS, thereafter incubated with Alexa Fluor 488 or 568 labelled chicken anti-rabbit IgG secondary antibody (1:200, Invitrogen, Life Technologies, Carlsbad, CA, USA) for 30 min at RT in the dark and then counterstained with Hoechst 33342 (1:2000, Sigma-Aldrich, USA) for 10 min. Finally, slides were coverslipped with Vectashield fluorescent mounting medium (Vector Laboratories, Burlingame, CA, USA). Appropriate controls were performed by omitting the primary antibodies to assure their specificity and to avoid autofluorescence. Fluorescence signals were analysed with a Nikon C2 confocal laser scanning microscope system (Nikon, Minato, Tokyo, Japan).

Human colon biopsies and mice bowel samples were embedded into Shandon cryomatrix (Thermo Fisher Scientific) and cut into 5 μm slides, stored at -80 °C until use. HT-29 and CCD-18Co cells were seeded in chambers and cultured for 24 h in 37 °C. After repeated washing slides were permeabilized with Cytofix/Cytoperm (BD) for 15 min at RT, then incubated with primary antibodies specific to αSMA (sc-53015; mouse, 1:2000, Santa Cruz Biotechnology, Dallas, TX, USA) or IL-20RB (ab124332; rabbit, 1:100, Abcam) for 1 h at RT. After repeated washing slides were incubated with Alexa Fluor 488 goat anti-mouse IgG (A11001, Invitrogen) and Alexa Fluor 568 donkey anti-rabbit secondary antibody (A10042, Invitrogen), both diluted to 1:100 for 30 min at RT in the dark and counterstained with Hoechst 33,342 (1:2000, Sigma-Aldrich). Finally, slides were rinsed in PBS and coverslipped with Vectashield fluorescent mounting medium (Vector Laboratories, Burlingame, CA, USA). Appropriate controls were performed omitting the primary antibodies to assure their specificity and to avoid autofluorescence. Sections were analyzed with a Nikon C2 confocal laser scanning microscope system (Nikon, Minato, Tokyo, Japan).