Cd3 no n1580

Quantitative in situ immunohistochemical analyses were performed by counting positively stained cells by blinded independent investigators microscopically in at least six high power fields (HPF, 0.287 mm², 400 × magnification) of tissue sections (see above) of intestinal ex vivo biopsies as recently reported [20 (link),57 (link)]. In brief, detection of apoptotic epithelial cells, macrophages and monocytes, T lymphocytes, regulatory T cells and B lymphocytes, 5 µm thin colonic paraffin sections were stained with primary antibodies directed against cleaved caspase3 (Asp175, Cell Signaling, Beverly, MA, USA, 1:200), F4/80 (no. 14-4801, clone BM8, eBioscience, San Diego, CA, USA, 1:50), CD3 (no. N1580, Dako, 1:10), FOXP3 (clone FJK-165, no. 14-5773, eBioscience, 1:100) and B220 (no. 14-0452-81, eBioscience; 1:200), respectively.

Apoptotic epithelial cells, macrophages and monocytes, T lymphocytes, regulatory T cells, and B lymphocytes were quantified following in situ immunohistochemical staining of 5 μm thin colonic paraffin with primary antibodies directed against cleaved caspase-3 (Asp175, Cell Signaling, Beverly, MA, USA, 1:200), F4/80 (no. 14-4801, clone BM8, eBioscience, San Diego, CA, USA, 1:50), CD3 (no. N1580, Dako, Glostrup, Denmark, 1:10), and FOXP3 (clone FJK-165, no. 14-5773, eBioscience, San Diego, CA, USA, 1:100), respectively, as reported earlier [40 (link),41 (link)]. The average number of respective positively stained cells in each sample was assessed within six high power fields (HPF, 0.287 mm², 400× magnification, light microscopy) by an independent and blinded investigator.

For in situ immunohistochemical analyses, colonic ex vivo biopsies were fixed in 5% formalin and embedded in paraffin as recently reported [62 (link)]. In brief, an independent investigator counted numbers of specifically stained cells by light microscopy in order to detect apoptotic epithelial cells, neutrophils, macrophages and monocytes, T lymphocytes, and regulatory T cells. Respective cells were counted in colonic paraffin sections (5 µm) stained with primary antibodies against cleaved caspase-3 (Asp175, Cell Signaling, Beverly, MA, USA, 1:200), MPO7 (No. A0398, Dako, Glostrup, Denmark, 1:500), F4/80 (no. 14-4801, clone BM8, eBioscience, San Diego, CA, USA, 1:50), CD3 (no. N1580, Dako, 1:10), and FOXP3 (clone FJK-165, no. 14-5773, eBioscience, 1:100), respectively. The mean number of positively stained cells in each blinded sample was determined within at least six high-power fields (HPF, 0.287 mm², 400 × magnification).