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All IHC slides, other than VEGF, were quantitated using the following protocol. The image was acquired using a Nikon DXM1200F Digital Camera (Nikon Eclipse 80i Microscope, Nikon ACT-1 v2.70 software) and analyzed using Image-Pro Plus v5.0 with custom-designed macros. Parameters for image analysis were as follows:
(i) All images were taken at a constant exposure associated with a particular group of stains.
(ii) Images were acquired pseudorandomly with no images overlapping the same areas.
(iii) All macros were constructed from a random set of images from the associated stain to compensate for variability within the group.
(iv) All images were acquired at ×20 (×200 total magnification).
(v) Ten areas were randomly taken for each slide, and data are presented as an average.
Special notes per stain are as follows. For PSA analysis, no post-capture modification was done to the images in order to preserve the color intensity of each sample. A 3-tier intensity scale was arbitrarily constructed to sort the varying degree of positive cells into groups. Data supplied included raw area in millimeter square and percent composition of each group of cells that make up the particular image and were expressed as PSA expression indices. For PCNA analysis, all images were calibrated to a master (single image) color profile to maximize accuracy.
Total cells were counted as well as total tissue area (excluding white space). Data were presented as cells per area of tissue in millimeter square and expressed as proliferation indices. For apoptosis analysis, all images were calibrated to a master (single image) color profile to maximize accuracy. Apoptotic cells were analyzed through contrast enhancement algorithms. Data were presented as raw apoptotic cell counts and expressed as apoptosis indices. For PECAM-1 analysis, all images were calibrated to a master (single image) color profile to maximize accuracy. Vessels were analyzed through contrast enhancement algorithms. Non-specific/background signal was filtered out via morphological algorithms. Data were presented as raw vessel counts and expressed as angiogenesis indices.
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