No 14 0452 81

In situ immunohistochemical analyses were performed in colonic ex vivo biopsies that had been immediately fixed in 5% formalin and embedded in paraffin as described earlier [44 (link)–46 (link)]. Paraffin sections (5 μm) of ex vivo biopsies from colon, liver and kidneys were stained with primary antibodies directed against cleaved caspase 3 (Asp175, Cell Signaling, Beverly, MA, USA, 1:200) for detection of apoptotic epithelial cells; against Ki67 (TEC3, Dako, Denmark, 1:100) for detection of proliferating epithelial cells; against F4/80 (# 14-4801, clone BM8, eBioscience, San Diego, CA, USA, 1:50) for detection of macrophages/monocytes; against CD3 (#N1580, Dako, 1:10) for detection of T lymphocytes; and against B220 (No. 14-0452-81, eBioscience; 1:200) for detection of B lymphocytes. Secondary antibodies were used for detection as previously described [31 (link), 47 (link)]. Positively stained cells were examined by light microscopy (magnification 100× and 400×), and for each mouse the average number of respective positively stained cells was determined within at least six high power fields (HPF, 0.287 mm², 400× magnification) by an independent investigator using blinded samples.

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.

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).