Angiogenin

The sections were deparaffinized and treated with Antigen Unmasking Solution (Vector, Burlingame, CA, USA) for 30 min at 100 °C. Subsequently, these sections were washed with phosphate buffered saline (PBS), and endogenous peroxidase activity was blocked by incubation in methanol containing 0.3% H₂O₂ for 30 min. After blocking with 2% horse serum (Vector) for half an hour, the sections were incubated overnight with primary antibodies against osteoprotegerin (Abcam, Cambridge, UK), alkaline phosphatase (GeneTex), angiogenin (Abcam), and osteopontin (Abcam). The sections were then incubated with biotinylated anti-rabbit IgG antibody (Vector) for 60 min. The ABC complex system (Vector) was used to subsequently develop peroxidase via incubation in a VIP solution (Vector) to visualize the peroxidase label. Images were obtained with a microscope equipped with a Charge-coupled Device (CCD) camera (AxioCam HRC, Zeiss, Jena, Germany) to detect the signal.

Immunohistochemistry was used to detect bronchial vascular biomarkers. The primary monoclonal antibodies against mouse transcription factor (ERG, 1:80), epidermal growth factor (EGF, 1:100), insulin-like growth factor (IGF-1, 1:400), endothelin-1 (1:600), angiogenin (1:400) and amphiregulin (1:500) were purchased from Abcam (Hong Kong, China). The primary monoclonal antibody against mouse Von Willebrand factor (vWF, 1:50) was purchased from CHEMICON International, Inc. MA 01730. The PAP (peroxidase anti-peroxidase) technique was used as previously described [13 (link)-15 (link)]. Positively staining cells were detected using the glucose oxidase-DAB-nickel method [19 (link)]. At least 8 random high-power fields (200 × total magnification) of each mice in the 3 groups should be independently analysed by 2 observers in a blinded fashion on a Leica DM6000B microscope (Leica, Wetzlar, Germany) connected with a Leica Application Suite Version 3.6. The data were expressed as percentages of positive stained areas per unit area of entire lung sections.