Immunohistochemical Staining of Colon Explants

Immunohistochemical stainings were done in large intestinal ex vivo explants, following immediate fixation of the tissues in 5% formalin and embedding in paraffin as recently reported [63 (link),64 (link),65 (link),66 (link)]. For enumerating apoptotic epithelial cells, 5 μm thin colonic paraffin sections were stained with primary antibodies directed against cleaved caspase 3 (Asp175, Cell Signaling, Beverly, MA, USA; 1:200); for proliferating epithelial cells against Ki67 (TEC3, Dako, Glostrup, Denmark; 1:100); for macrophages/monocytes against F4/80 (# 14-4801, clone BM8, eBioscience, San Diego, CA, USA; 1:50); for T lymphocytes against CD3 (#N1580, Dako; 1:10); for regulatory T cells (Tregs) against FOXP3 (clone FJK-165, #14-5773, eBioscience; 1:100); and for B lymphocytes against B220 (No. 14-0452-81, eBioscience; 1:200). Positively stained cells were enumerated by an independent investigator applying light microscopy. The average number of respective positively stained cells in each sample was determined within at least six high power fields (HPF, 0.287 mm², 400× magnification).