Quantification of TKR immunopositivity in normal and tumor TMA cores using algorithm analysis software. (A) Positive pixel analysis is a digitalized pixel count in a user defined region (red square) of each core as shown in the visual representation of the TMA core for PDGFRα analysis (right). The bar graph represents the percent strong positive pixels for each TKR and VEGF (left). The fraction of strong stained pixels is the number of total pixels (blue and red) minus the negative pixels (blue) in the markup image of the TMA, divided by the number of total stained pixels. (B) Color deconvolution analyses of IHC digitalized images accounted for different staining densities of the entire core. The intensity range markup core of PDGFRα analysis (right) shows intensity ranges as weak positive staining (yellow), medium (orange), strong positive (red) and negative staining (blue). The biospecimen percent of positive pixels strongly stained for TKRs and VEGF are represented in the bar graph on the left. Each bar represents the mean positivity of 36 tumor (black) and 36 normal (grey) TMA cores from 12 dogs. Error bars, mean ± SEM. *cpAC vs. normal: P < .05.