Quantitative Analysis of Intestinal Tissue

Intestinal tissues were fixed in 4% paraformaldehyde (Sigma-Aldrich) dissolved in phosphate-buffered saline (PBS), embedded in paraffin, and sectioned to a thickness of 3.5 μm. H&E and IHC staining for β-catenin and Ki67 were performed as previously described [3 (link)]. The sections were scanned using a NanoZoomer 2.0-HT Digital slide scanner C9600 (Hamamatsu) and analyzed with NDP.view 2 software. Ki67 positive proliferating cells were quantified as the percentage diaminobenzidine (DAB) positive area per total hematoxylin stained nuclear area, using ImmunoRatio, a publicly available application for quantitative image analysis [33 (link)]. Activated EGFR levels were assessed as described previously [3 (link)], using an antibody against EGFR phosphorylated on Tyr1068 and Alexa Fluor 488 conjugated secondary anti-rabbit antibody, and measuring the fluorescence intensity along a line across colonic crypts. Apoptotic intestinal epithelial cells were detected using the ApopTag^® Peroxidase In Situ Oligo Ligation (ISOL) Apoptosis Detection Kit as recommended by the manufacturer (Chemicon), and quantified using Axiovision Software (Zeiss) and manual counting of stained cells per mm² tissue from 3 randomly chosen mucosal areas. For quantifications, colon sections from 4-5 controls and 4-5 Pacs2^-/- mice were used.