Histopathology, immunohistochemistry (IHC) and immunofluorescence
Colon biopsies for histopathology were fixed overnight in 10% formalin. After paraffin embedding, 3-μm sections were cut and stained with hematoxylin and eosin (H&E) in accordance with standard protocols for microscopic examination. IHC was performed on 3 μm sections of paraffin-embedded fresh-frozen endoscopic colon tissues from patients with IBD and control individuals. The sections were deparaffinized, and antigen was retrieved by microwave oven irradiation using sodium citrate buffer, followed by blocking with 5% goat serum for 30 minutes at room temperature. The sections were incubated with the primary antibody overnight at 4°C. After careful washing, the sections were then incubated for 1 hour at room temperature with a secondary antibody. Sections treated with phosphate-buffered saline (PBS) instead of the primary antibody were used as a negative control. Images were acquired by a microscope (Olympus, Cat. #IX73). Image-Pro Plus version 6.0 (Media Cybernetics, BetheSEMa, MD) was used to calculate the integrated option density (IOD).