2.5 Immunohistochemistry analysis of intestinal tissues.
Immunohistochemistry analysis was conducted to evaluate the expression of relative proteins. The paraffin-embedded tissue sections were deparaffinized and treated with hydrogen peroxide (3 m/v) for 15 min to remove endogenous peroxidase. Antigen retrieval was performed by blocking the samples in goat serum for 10 min at 22 °C. The following antibodies were added and incubated for 12 hours at 4 °C: anti-TLR4 antibody (Servicebio, GB11519, 1:500), anti-LC3B antibody (Abcam, ab86714, 1:500), and anti-NF-κB p65 antibody (Servicebio, GB13025-1, 1:500). Cy3 (Servicebio, GB21303) and FITC (Servicebio, GB22303) secondary antibodies (1:250) were used for visualization. Tissue sections were mounted and analyzed using an inverted fluorescence microscope (Nikon Eclipse TI-SR) and imaging system (Nikon DS-U3).