Automated quantitative pathology system

For histological analysis, duodenum, jejunum, ileum and colon tissues from WT and EpCAM^−/− mice were fixed overnight with 4% paraformaldehyde in PBS at 4°C before being dehydrated and embedded in paraffin. Then, hematoxylin and eosin (H&E) staining was performed on 4-µm paraffin sections; the sections were incubated in hematoxylin for 2 min and eosin for 30 sec at room temperature. Images were captured using the PerkinElmer Automated Quantitative Pathology System.

For immunofluorescence staining, duodenum, jejunum, ileum and colon tissues from WT and EpCAM^−/− mice were fixed overnight with 4% para-formaldehyde in PBS at 4°C before being dehydrated and embedded in optimal cutting temperature compound (OCT) (Sakura Finetek). Then, 7-µm frozen sections were boiled in 10 mM citric acid (Merck) at pH 6.0 for 5 min. After exposure to goat serum blocking buffer (ZSGB-BIO, ZLI-9056) at room temperature for 1 h, the sections were incubated overnight at 4°C with primary antibodies and then with secondary antibodies at room temperature for 1 h. Primary antibodies were used as follows: Rabbit anti-E-cadherin (Cell Signaling Technology, Inc.; cat. no. 14472, 1:200), rabbit anti-p120-catenin (Santa Cruz Biotechnology, Inc.; cat. no. 15D2, 1:200), and rabbit anti-β-catenin (BD Biosciences; cat. no. 610154, 1:200). Immunofluorescence analysis was performed with Alexa Fluor 488-labeled donkey anti-rabbit IgG (H+L) secondary antibodies (Thermo Fisher Scientific, Inc.; cat. no. A21206, 1:1,000), and DAPI (Sigma-Aldrich; Merck KGaA; cat. no. D9564, 1:10,000) was used to stain the nuclei of tissues. Immunofluorescence images were observed using a PerkinElmer Automated Quantitative Pathology System.