Fig. 1. Small ICAM-1 immunoconjugates are internalized by HUVEC. HUVEC were treated with 250 U TNF- for 24 hours. Confluent monolayers were incubated at either 4°C (c) or 37°C (A,C-F) in the presence of either large (A; >1000 nm diameter), small (b; <500 nm diameter) biotin-anti-ICAM-1/streptavidin conjugates, anti-ICAM-1 immunobeads (C,D) or beads previously coated with control murine IgG (E,F). The cells were subsequently washed, fixed and counterstained with fluorescent goat anti-mouse IgG. Merged images corresponding to representative samples were pseudocolored to show single-labeled, internalized immunoconjugates/immunobeads as green (arrows) and double-labeled immunoconjugates/immunobeads on the cell surface as yellow (arrowheads). The phase-contrast image shown in e corresponds to the fluorescence image shown in F. Bar, 10 µm. (G) Uptake of anti-ICAM-1 (l) and anti-PECAM-1 (°) immunobeads by TNF--activated HUVEC was determined for different incubation times as the mean percentage of internalized (single labeled) immunobeads per cell. Error bars corresponding to s.d. were smaller than the size of the symbols used for the graph.