Immunohistochemical analysis (NF-κB p65)

Immuno-histochemical (IHC) staining was conducted using the streptavidin-biotin complex (strept-ABC) method (Wuhan Boster Biological Technology, Ltd.). Unstained 4-mm sections were cut from paraffin blocks and incubated at 65°C for 30 min. The slides were deparaffinized in xylene followed by absolute ethanol and subsequent rehydration in graded ethanol. Antigen retrieval was performed by immersing slides in boiling citric acid buffer (pH 6.0) for 15 min. The sections were then pretreated with 3% hydrogen peroxide for 15 min to inactivate endogenous peroxides, followed by incubation with 10% goat serum (Sigma-Aldrich; Merck KGaA) at 37°C for 30 min to block the non-specific binding. Slides were incubated overnight at 4°C with anti-NF-κB p65 antibody (3301; 1:100; Wuhan Boster Biological Technology, Ltd.), subsequently washed with PBS before applying the biotinylated secondary antibody (SE587; Sigma-Aldrich; Merck KGaA) at 37°C for 10 min. Sections were incubated with the strept-ABC complex reagent for 15 min, and subsequently exposed to 3,3′-diaminobenzidine, counterstained with hematoxylin and examined by light microscopy.

The degree of stained sections was scored in duplicate by two independent investigators without any histopathological information. IHC scores were determined by combining the proportion of positively stained cells and intensity of staining in five different high power fields (x400) for each section. Staining intensity was graded as follows: 0, no staining; 1, weak staining (light yellow); 2, moderate staining (yellowish brown); and 3, strong staining (brown). The percentage of positive cells was scored according to the following criteria: 0, no positive cells; 1, ≤25% of cells stained positive; 2, 26–50% of cells stained positive; 3, 51–75% of cells stained positive; and iv) >75% of cells stained positive.