Immunohistochemical Analysis of Colonic Tissue

Colons were excised and cleaned with phosphate buffered saline followed by fixation in neutral buffered formalin (Thermo Fisher, Waltham, MA, USA). The fixed colon tissues were embedded in paraffin and 5 μm thick sections were sliced and placed on glass microscope slides. Immunohistochemical staining was performed as described previously.^{8 (link)} Briefly, sections were deparaffinized, followed by antigen retrieval using the antigen retrieval solution (Agilent Technologies, Santa Clara, CA, USA). Endogenous peroxidase activity was quenched with 3% H₂O₂ in phosphate buffered saline for 10 min. Sections were stained using specific primary antibodies and Vectastain ABC kit and diaminobenzidine (Vector Laboratories, Burlingame, CA, USA). Counterstaining was performed with hematoxylin and stained sections were visualized by Leica DM550B microscope. Alternatively, in some experiments, fluorescent dye-labeled secondary antibody was used and sections were visualized by LSM 510 (Zeiss) confocal microscopy.