Immunofluorescent studies on COS.TM-CR cells. Cells were preincubated on ice for 45 minutes with 24FM, washed, and placed at 37°C for 0, 10, and 30 minutes (A, B, and C, respectively). After permeabilization and fixation, antibody localization was detected using FITC as described in the Materials and Methods. In (B) and (C), antibody-TM complex was present intracellularly in clusters, with less on the plasma membrane as compared with (A). (D) After incubation at 37°C for 30 minutes as in (C), the cells were fixed without permeabilization. The intensity of labeled TM at the cell surface was reduced, indicating that constitutive internalization of the receptor had occurred.