Immunohistochemical staining was performed on 5-μm para n sections by using immunoperoxidase visualization. After routine depara nization, heat-induced epitope retrieval was performed through immersion of the slides in 0.01 M sodium citrate buffer (pH 6.0). To block the endogenous peroxidase activity and nonspeci c binding of antibodies, the sections were preincubated for 1 h at room temperature in 0.1 M PBS containing 10% normal goat serum and 0.3% H 2 O 2 . The sections were then incubated for 20 h at 4°C with rabbit polyclonal anti-vWF antibody (1:100; Abcam, Cambridge, Massachusetts, USA) or rabbit polyclonal anti-VEGF antibody (1:50; Santa Cruz Biotechnology) as primary antibodies. The sections were then treated for 1 h at 37°C with biotinylated goat antimouse or antirabbit IgG (1:200, Jackson ImmunoResesarch Laboratories Inc.). Following the reaction produced using reagents from an avidin-biotin complex kit (Vector Laboratories, Inc.), the reaction products were visualized using a diaminobenzidine substrate kit (Vector Laboratories, Inc.) according to the recommendations of the manufacturer.
Lung cytokines IL-1β, TNF-α, and MIP-2 levels were determined using the Bio-Plex multiplex assay system (Bio-Rad, Hercules, CA, USA) and Procarta immunoassay kit (Affymetrix) according to the manufacturers' protocols.
Lung cytokines IL-1β, TNF-α, and MIP-2 levels were determined using the Bio-Plex multiplex assay system (Bio-Rad, Hercules, CA, USA) and Procarta immunoassay kit (Affymetrix) according to the manufacturers' protocols.