Neu5Gc in the endothelium and subendothelium of atherosclerotic plaques. (A) Expression of Neu5Gc in endothelium overlying human aortic atherosclerotic plaques is shown by double labeling with the cGcAb anti-Neu5Gc antibody (left) the endothelial marker CD31 (center, see merge, right). White arrows indicate subendothelial Neu5Gc within lesions (left). (B) Samples of human aortic atherosclerotic plaques were identified by their characteristic appearance (hematoxylin and eosin, bottom right). Accumulation of macrophages (stained with anti-CD68, top middle) and oxidized-LDL (stained with malondialdehyde, top right) are shown. The cGcAb (top left) was used to detect the presence of Neu5Gc on the endothelium of the plaques. Control IgY and mouse IgG (bottom right and middle) demonstrates specificity of the staining. (C) Human aortic atherosclerotic plaque sections were double-stained with anti-CD68 (middle) for macrophages and cGcAb (left) for Neu5Gc. The merged fluorescent image (right) indicates that macrophages are recruited to the Neu5Gc-lined endothelium. (D) Endothelium was labeled with anti-VWF antibody (center; note that there is also some subendothelial green auto-fluorescence caused by macrophages and/or necrotic foci). In addition to typical endothelial-colocalized Neu5Gc staining, extensive subendothelial labeling with the cGcAb anti-Neu5Gc antibody (red channel, left) is seen in this lesion (see yellow in the merged image, right). All pictures in this figure are representative examples of multiple independent analyses on multiple samples from multiple individuals.