Histological Analysis of Intestinal Tissue

For histological analysis, longitudinal sections from the large intestine were immediately fixed according to previously described protocols [8 (link)]. Colon sections with a thickness of 5 μm were stained with hematoxylin and eosin (H&E) to visualize morphology or with alcian blue to visualize goblet cells using an optical microscope (Axio Vert.A1, Carl Zeiss, Oberkochen, Germany). For immunohistochemical and immunofluorescence staining, sections were incubated overnight at 4 °C with primary antibodies against p-STAT3, Ly6G (both from GeneTex, Irvine, CA, USA), iNOS, Arginase-1 (both from Cell Signaling Technology, Danvers, MA, USA) and then developed following a conventional technique. Samples were analyzed with a ZeissVert.A1 conventional epifluorescence microscope and a LEICA TCS SP8X confocal microscope.