Proximal, mid and distal segments of the SI were fixed with 4% paraformaldehyde (PFA) from 3 h to overnight at 4 °C, embedded in OCT and kept at −80 °C until sectioning. Sections (7 µm thick) were prepared with a cryotome (Cryostat CM 3050S).
Images from Hematoxyline QS (Vector) stained were acquired using NDP Zoomer Digital Pathology (Hamamatsu) and subsequently analysed in NDP.view2 software.
To perform immunofluorescence, sections were blocked and permeabilized in 10% adult bovine serum (Sigma), 5% skim milk (Sigma) and 0.3% Triton X-100 in PBS (blocking buffer) for at least 1 h at 4 °C. Primary antibodies (listed in Supplementary Table3 ) were incubated overnight in blocking buffer at 4 °C. Alexa-Fluor-Conjugated secondary antibodies (indicated in Supplementary Table 3 ) were incubated for 1–2 h at room temperature in 10% adult bovine serum and 0.1% bovine serum albumin (BSA) (Sigma) in PBS. Diamidino-2-phenylindole dihydrochloride (DAPI; 1 µM; Sigma) was used to counterstain nuclei in the indicated experiments. Images were acquired using laser-scanning confocal microscopes (Leica TSC SP8 and Zeiss LSM800). All images were subsequently analysed with Fiji software.
Images from Hematoxyline QS (Vector) stained were acquired using NDP Zoomer Digital Pathology (Hamamatsu) and subsequently analysed in NDP.view2 software.
To perform immunofluorescence, sections were blocked and permeabilized in 10% adult bovine serum (Sigma), 5% skim milk (Sigma) and 0.3% Triton X-100 in PBS (blocking buffer) for at least 1 h at 4 °C. Primary antibodies (listed in Supplementary Table