IGF-II binding and internalization via IR by confocal microscopy. A. HeLa cells expressing IR-B-SYFP were labeled in vivo with 50 nM IGF-II-biot and 2 nM QD655 at room temperature and fixed in 3.7% PFA. Lower panel shows cells incubated at 37°C for 150 min after labeling and treated with acid (0.5 M NaCl, 0.1 M Na-glycine pH 3) for 5 min before fixation. Imaging was performed by confocal microscopy (Olympus Fluoview FV 1000). Scale bars: 10 μm. B. HeLa cells expressing IR-B-GFP were labeled in vivo with 50 nM IGF-II-biot and 2 nM QD655, incubated for 1 hour at 37°C, then with 0.1 mM Na-glycine pH 3, NaCl 0.5 M for 5 min and fixed in 3.7% PFA. Images were taken with a confocal microscope (Olympus Fluoview FV 1000) by acquisition of 64 z-slices with 16 μm step size. Deconvolution was performed with Huygens Scientific Volume Imaging Huygens Professional Version 3.6, applying the Quick maximum likelihood estimation algorithm. Tridimensional reconstructions are shown. C. Maximum intensity projection. D. Surface renderer. Different views are shown. Lower panels show views of 14 z-slices section (2.24 μm) that is shown in the right lower corner of the third panel. Scale bars: 5 μm. E. Quantification of the degree of internalization of insulin and IGF-II (ratio between QDinterior and QDtotal). The results are shown as the mean ± s.e.m (n = 19–29 cells). Asterisks indicate significant differences between ligands (p ≤ 0.02). In A and B white arrows indicate internalized QD655 and orange arrows label non transfected cells.