After mice were sacrificed, the left lung lobe was dissected and fixed with 4% paraformaldehyde at room temperature for 48 h, dehydrated, embedded in paraffin and then sliced into 5-µm sections. The tissue sections were then placed in citrate buffer for antigen retrieval. After being boiled three times (5 min each), the sections were blocked with 3% H2O2 and incubated for 10 min to eliminate the internal peroxidase activity at room temperature. CRP primary antibody (1:500; cat. no. ab211631; Abcam) was added to the sections for 2 h at room temperature and the sections were then incubated with an HRP-labeled secondary antibody (1:1,000; cat. no. sc-2004; Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. The sections were exposed to DAB in the dark for 6 min and counterstained with hematoxylin for 10 min at room temperature, then dehydrated and sealed by neutral gum. Eight randomly selected sections from mice in each group were assessed. The expression of CRP in the lung tissue was observed under a light microscope. The optical density values were analyzed and measured using Image-Pro Plus 6.0 software (Media Cybernetics, Inc.).

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