In some experiments, mice selleck chemicals were injected intraperitoneally with soluble gp130-FC fusion protein (sgp130Fc, 0.5 mg/kg; R&D Systems, Minneapolis, MN, USA) or phosphate-buffered saline (PBS). All surgical and experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) in College of Medicine, Seoul National University. Isolation of Colonic Epithelial Cells CECs were isolated using a modified version of previously described methods [60], [61]. In brief, colon pieces were incubated in Ca2+- and Mg2+-free Hanks Balanced Salt Solution (HBSS; Sigma Chemical) containing 2% calf serum at 37��C for 30 min while stirring. After collecting the supernatant, remaining tissue was resuspended in HBSS with 3 mM ethylenediaminetetraacetic acid (EDTA).
The tissues were incubated while stirring at 37��C for 30 min. The supernatant was then collected and centrifuged at 300��g for 10 min. Histopathology and Immunohistochemistry of Colon Tissue Large intestine specimens were fixed in 10% neutralized formalin for 24 h, embedded in paraffin, and sectioned at 4 ��m. Colon sections were stained with hematoxylin and eosin (H&E) for histopathological analysis, and stained immunohistochemically as described previously to investigate the expression of phospho-STAT3 (STAT3PY705) [62]. Briefly, paraffin-embedded slides were hydrated with ethanol and distilled water, followed by antigen retrieval in 10 mM sodium citrate buffer. Sections were incubated with anti-S100A9 (Abcam, Cambridge, MA, USA) and anti-STAT3PY705 immunoglobulin G (IgG; Cell Signaling Technology, Danvers, MA, USA) and stained with 3,3-diaminobenzidine substrate.
Image acquisition and processing were performed using a Leica microscope and the Leica Application Suite (Leica Microsystems, Buffalo Grove, IL, USA). Immunofluorescence of Colon Tissue To perform immunofluorescence analyses, mouse large intestine specimens were embedded in optimal cutting temperature compound (Sakura Finetek Japan, Tokyo, Japan) and sectioned at 10 ��m by cryostats (Leica Microsystems). The sections were incubated with a combination of anti-Gr-1-biotin and purified anti-CD11c antibodies (BD Biosciences) or with rabbit anti-STAT3PY705 IgG at 4��C overnight, followed by incubation with appropriate secondary antibodies. Stained sections were mounted in VectaShield 4��, Entinostat 6-diamidino-2-phenylindole (DAPI) mounting medium (Vector Laboratories, Burlingame, CA, USA) and analyzed under a confocal laser scanning microscope (LSM 510; Carl Zeiss, Gottingen, Germany). Images were taken under a confocal microscope using a 40�� objective.