4 6 diamidine 2 phenylindole dapi

After the sacrifice of piglets, samples of proximal duodenum were immediately excised and fixed in 4% paraformaldehyde (Sigma-Aldrich) in phosphate-buffered saline (PBS) (Sigma) at 4°C for 24 h. After washing 3 times for 30 minutes in PBS, the fixed samples were successively soaked in 12.5 and 25% sucrose (Merck) for 1.5 and 12 hours, respectively at 4°C. The tissue was then embedded in Tissue-Tek compound, frozen in liquid nitrogen and sectioned into 20-μm slices using a cryostat (Leica). The sections were washed in PBS and permeabilized by bathing in PBS/0.1% Triton X-100 (Sigma) for 10 minutes. Non-specific antibody binding was blocked by incubation of the sections in PBS/3% BSA (Merck) for 1.5 hours. For protein detection, sections were incubated at RT with primary antibody (S3 Table) diluted in PBS/3% BSA. The sections were then washed 3 times with PBS and incubated with Cy3 (indocarbocyanine)-conjugated secondary antibody (S3 Table) diluted in PBS/3% BSA. Finally, the sections were washed 3 times for 10 minutes in PBS at RT and mounted using Vectashield with 4′,6-diamidine-2-phenylindole (DAPI; Vector Labs). As a negative control, some sections were prepared without incubating with primary antibody. IF was analyzed with a Zeiss LSM 510 Meta confocal microscope (Carl Zeiss, Jena, Germany) using the 60x objective.