Fig. 3. Anti-ICAM-1 immunobeads do not colocalize with caveolin or clathrin. TNF--stimulated HUVEC were incubated with control anti-ICAM-1 immunobeads (A,C) or Alexa 594-conjugated cholera toxin B subunit (B) for 15 minutes at 37°C. The cells were then washed and fixed, and surface-bound material was counterstained with TxR goat anti-mouse IgG (A,C) or goat anti-cholera toxin followed by fluorescein rabbit anti-goat IgG (B). After permeabilization, the cells were then labeled with rabbit anti-human caveolin-followed by Alexa 350-conjugated goat anti-rabbit IgG (A,B) or TRITC-conjugated anti-clathrin heavy chain (C). Insets show images magnified twofold. The image color channels were selected to facilitate the comparison between panels in the figure, and they are: green, internalized immunobeads or cholera toxin; blue, surface-bound immunobeads or cholera toxin; red, caveolin-1 (arrowheads) or clathrin (arrows). There was little, if any, colocalization of anti-ICAM-1 immunobeads with caveolin-1 or clathrin, as evidenced by the lack of yellow labeling in A and C and areas showing internalized immunobeads with little caveolin-1 or clathrin nearby (see insets).