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Fig. 3. I/F8-induced internalization of the ALCAM/CD166 molecule. IGROV-I cells were incubated with I/F8 scFv at 4°C in the presence (A,B,D) or in the absence (C) of the anti-FLAG-tag mAb as dimerizing agent. Cells were then placed on ice (A,B) or moved to 37°C for 30 minutes to induce internalization (C,D), and were subsequently untreated (A) or acid-washed to strip the surface-bound molecules (B-D). Samples were then fixed, permeabilized, stained with FITC-labeled secondary antibodies and visualized by using an Olympus confocal laser microscope system. Insets show staining with the irrelevant anti-NIP scFv as a negative control, except B where the Propidium Iodide-counterstained nuclei present in the same field are visualized. The residual staining detected in the stripped samples therefore corresponds to the endocytosed molecules. Magnification, 400 x.
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