Quantification of Pulmonary Fibrosis Markers

Transforming growth factor β (TGF-β), an important profibrotic growth factor and vascular endothelial growth factor (VEGF), a key angiogenic mediator of pulmonary fibrosis were determined by immunohistochemistry in lung tissues as previously described [33 (link)]. Lung sections were incubated overnight with anti-TGF-β (Santa Cruz Biotechnology; 1:100 sc-130348 in PBS, v/v, MA, USA) and anti-VEGF polyclonal antibody (Santa Cruz Biotechnology; 1:100 sc-7269 in PBS, v/v, MA, USA). At the end of the incubation with the primary antibodies, the sections were washed with PBS and incubated with a secondary antibody (Santa Cruz Biotechnology, Dallas, TX, USA) for 1 h at room temperature. The reaction was revealed by a chromogenic substrate (brown DAB), and counterstaining with nuclear fast-red. Images were collected using a microscope and AxioVision software. For graphical display of densitometric analyses, % positive staining (brown staining) was measured by computer-assisted color image analysis (Nikon Eclipse Ci-L microscope). The percentage area of immunoreactivity (determined by the number of positive pixels) was expressed as a percentage (%) of the total tissue area (red staining) at 20× magnification.