Quantifying VEGF and CD34 Expression in Lung Tissues

Immunohistochemistry (IHC) staining for vascular endothelial growth factor (VEGF) and CD34 was measured in the lung tissues as previously described [62 (link)]. Briefly, lung sections (7 μm) were processed and incubated overnight with anti-VEGF polyclonal antibody (Santa Cruz Biotechnology; 1:500 #sc7269 in PBS, v/v, MA, USA) and anti-Caspase-3 (Santa Cruz Biotechnology; 1:500 #sc7272 in PBS, v/v, MA, USA). To verify antibody-binding specificity, control slices were incubated with only primary antibody or secondary antibody, neither of which gave positive staining. Images were collected using a Zeiss microscope and Axio Vision software. For graphic display of densitometric analyses, the % of positive staining (brown staining) was measured by computer-assisted color image analysis (Leica QWin V3, UK). The percentage area of immunoreactivity (determined by the number of positive pixels) was expressed as % of total tissue area (red staining) within five random fields at ×40 magnification.