The distribution of cell-surface nucleolin in vitro and in vivo. (A) Colocalizations between ES (green) and NL (red) on HMECs with different growth states were detected by indirect immunofluorescence. DAPI (blue) indicates cells in the field. Scale bar represents 20 μm. (B) HMECs with different growth states were incubated with ES for 1 hour, and then applied to co-IP and IB with anti-NL and anti-ES antibody, respectively. Actin blot serves as loading control. (C) Biotinylated ES and the anti-NL antibody were simultaneously injected intravenously into mice bearing B16/F10 tumors. Biotinylated ES and nonimmune rabbit IgG were simultaneously injected intravenously as control tumor. After 1 hour, the distribution of biotinylated ES (red) and anti-NL antibody (green) in heart, liver, kidney, lung, tumor, and control tumor was detected with TRITC-conjugated streptavidin and FITC-conjugated goat anti-rabbit IgG, respectively. DAPI (blue) indicates cells in the field. Arrows indicate the blood vessels in the field. Inset boxes show the magnified photos of the blood vessels in normal tissues. Scale bar represents 50 μm in the sections of heart, liver, kidney, and lung, and 20 μm in the sections of tumor, control tumor, and inset boxes. (D) Variant expression levels of cell-surface-NL in 2 colorectal tumor cases. The sections of primary colorectal carcinomas were stained with anti-CD31 (green) and the cell-surface NL–specific antibody (red). Scale bar represents 50 μm. The magnified pictures were shown in lower panels of each case. Scale bar represents 20 μm.