The immunohistochemistry technique was used for the levels of NF-κB, IL-6, TNF-α, and IL-1β expression according to the methodology described previously (23 (link)). Paraffin sections were deparaffinized and underwent antigen repairing. Then, sections were incubated with the primary antibody (1:50) at room temperature for 1-2 hours. Subsequently, sections were incubated with the secondary antibody. Then, they were visualized by incubating them with 3, 3'-diaminobenzidine etrahydrochloride (DAB) (Servicebio, China). The primary antibodies were the following: IL-1β polyclonal antibody (Bioworld Technology, co. Ltd., China; Cat.no. BS6067; rabbit), anti-IL-6 antibody (Boster, China; Cat. no. BA4339; rabbit IgG), TNF-α antibody (Affinity Biosciences, China; Cat. no. AF7014; rabbit polyclonal), and anti-NF-κBp65 antibody (Boster, China; Cat. no. BA0610; rabbit IgG).
The sections were evaluated with a high-power objective lens (25×) using optical microscopy (E100 LED, Nikon Corporation, Japan). The immunostaining intensity of pro-inflammation was evaluated by two examiners under double-blinded conditions. Possible scores were as follows. Score 1: 0% of positive cells. Score 2: <10% of positive cells or isolated cells. Score 3: 11–50% of positive cells. Score 4: >50% of positive cells (25 (link)).
The sections were evaluated with a high-power objective lens (25×) using optical microscopy (E100 LED, Nikon Corporation, Japan). The immunostaining intensity of pro-inflammation was evaluated by two examiners under double-blinded conditions. Possible scores were as follows. Score 1: 0% of positive cells. Score 2: <10% of positive cells or isolated cells. Score 3: 11–50% of positive cells. Score 4: >50% of positive cells (25 (link)).