Representative colocalization patterns of GLUT-1, CA IX, CD34 and Ki67 in an invasive squamous cell carcinoma of the vulva. GLUT-1 (upper left panel) is expressed preferentially in the outer layers of the tumor cell aggregates, while CA IX (upper right panel) is expressed at a distance from the stroma, which contains CD34-positive tumor microvessels (middle left panel). The lower left panel shows a merged image of thresholded positive pixels of GLUT-1 (green), CA IX (red) and CD34 (blue) and illustrates minimal overlap (yellow) between GLUT-1 and CA IX. It also underscores the aforementioned spatial relationships of GLUT-1 and CA IX with CD34-positive microvessels (blue). Ki67-positive proliferating cells (middle right panel) are found in the same compartment as GLUT-1 positive cells. Conversely, the expression of Ki67 and CA IX is virtually mutually exclusive. The lower right panel shows a merged image of thresholded positive pixels of GLUT-1 (green), CA IX (red) and Ki67 (blue). Any overlap between Ki67 and GLUT-1 and Ki67 and CA IX is visible in cyan and pink colors, respectively. All panels: cropped frames from 10x magnification digital images.