Histopathology and immunohistochemistry (IHC)

Formalin-fixed, paraffin-embedded, 5 µm thick intestine sections were used for histopathology by standard hematoxylin and eosin (H&E) staining method and IHC staining. The sections were randomly selected from jejunum and ileal sections of the small intestine. Deparaffinization of the sections followed by heat based epitope retrieval and both endogenous peroxidase blocking (3% H₂O₂, 5 min) and serum blocking (5% normal serum, 1 h) were carried out. Further, sections were incubated with primary antibody (1:500) for 2 h at RT and then species-specific anti-IgG secondary antibodies and conjugation with HRP was performed using Vectastain Elite ABC kit following manufacturer’s protocols. Finally, 3, 3-Diaminobenzidine (DAB) were used as a chromogen substrate and counter stained with Mayer’s hematoxylin. Morphometric analysis was done using CellSens Software from Olympus America.