Immunohistochemical Analysis of Colonic Biopsies

In situ immunohistochemical analyses were carried out in colonic ex vivo biopsies immediately fixed in 5% formalin and embedded in paraffin, as previously described [41 (link),48 (link),49 (link),50 (link)]. For detection of apoptotic and proliferating epitshelial cells, macrophages/monocytes, T lymphocytes, regulatory T cells, and B lymphocytes, 5 μm thin colonic paraffin sections were stained with primary antibodies directed against cleaved caspase 3 (Asp175, Cell Signaling, Beverly, MA, USA, 1:200), Ki67 (TEC3, Dako, Glostrup, Denmark, 1:100), F4/80 (# 14-4801, clone BM8, eBioscience, San Diego, CA, USA, 1:50), CD3 (#N1580, Dako, 1:10), FOXP3 (clone FJK-165, #14-5773, eBioscience, 1:100), and B220 (No. 14-0452-81, eBioscience; 1:200), respectively. Positively stained cells were enumerated using light microscopy, and the average number within at least 6 high-power fields (HPF, 0.287 mm², 400× magnification) was recorded by an unbiased investigator.