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).