Rabbit anti il 4

Immunohistochemical staining was done to compare changes in inflammatory cytokines between the groups. Briefly, the prepared sections were incubated in a 0.3% hydrogen peroxide (H₂O₂) solution and immersed in a 10% normal donkey serum (in 0.1M PBS, pH 7.4) solution for 30 min, respectively, at room temperature, as previously described [28 (link)]. Next, they were washed with PBS and immunoreacted with each primary antibody overnight at 4 °C. The primary antibodies were (1) and (2) rabbit anti-TNF-α (1:1000; Abcam, Cambridge, UK) and rabbit anti-IL-1β (1:200; Abcam, Cambridge, UK) for pro-inflammatory cytokines, (3) and (4) rabbit anti-IL-4 (1:250; Santa Cruz Biotechnology, CA, USA) and, mouse anti-IL-13 (1:250; Santa Cruz Biotechnology, CA, USA) for anti-inflammatory cytokines, and (5) rabbit anti-HMGB1 (1:1000; Abcam, Cambridge, UK) for inflammatory mediator. These immunoreacted sections were subsequently reacted with secondary antibody (biotinylated anti-rabbit IgG; 1:200; Vector, CA, USA) for 2 h at room temperature and immersed in an avidin-biotin complex (1:250; Vector, CA, USA) for 1 h at room temperature. Finally, these sections were colorized in brown with 0.05% 3, 3′-diaminobenzidine tetrahydrochloride (Sigma–Aldrich, St. Louis, MO, USA) solution (in 0.1 M PBS).