To evaluate ICAM-1, VCAM-1 and iNOS expression in lung tissue, immunohistochemistry was performed. Lung tissues were fixed in 4% paraformaldehyde at 4°C for 2 h and embedded in paraffin at 60°C for 2 h. Tissues were then blocked with 10% BSA (Sigma-Aldrich, Inc.) at 25°C for 30 min. Subsequently, the sections (thickness, 3-5 µm) were incubated with anti-ICAM-1 (cat. no. p05362), anti-VCAM-1 (cat. no. p29533) and anti-iNOS (cat. no. p35228) antibodies (all 1:800) overnight at 4°C. Following primary antibody incubation, the samples were incubated with an anti-rabbit IgG secondary antibody (cat. no. 3678s; 1:1,000) at 25°C for 30 min. The sections were stained with 0.05% DAB at 25°C for 5 min. Immunohistological analysis was performed using a fluorescence microscope (magnification, ×1,000). The optical density (OD) of the positively stained area was quantified using Image-Pro Plus software (version 6; Media Cybernetics Inc.).

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