Immunofluorescent studies on COSdel.238 cells. Cells were preincubated on ice for 45 minutes with 24FM, washed, and exposed to 37°C for 0, 10, and 30 minutes (A, B, and C, respectively). After permeabilization and fixation, antibody localization was detected using FITC conjugated to a secondary antibody. In representative fields as shown in (A), (B), and (C), antibody-TM complex was seen predominantly on the cell surface, with little evidence of internalization or significant shedding of the complex from the cell surface over 30 minutes. In (D), cells were prepared as in (C), but with murine monoclonal anti-transferrin receptor antibodies CD71. In this case, intracellular clusters of antibody-receptor complexes were observed, indicating that the cells were capable of supporting constitutive endocytosis.