Imaging of biotinylated ligands binding and internalization by QDs. A. Labeling strategy. B. QD655 concentration curve: HeLa cells expressing IR-B-GFP were labeled with 50 nM BAC-Ins for 15 min and different concentration of QD655 (0.5 nM to 4.0 nM) for 10 min at 15°C. Quantification of the ratio between QD655 and GFP signal (GFP/QD655). Asterisks indicate significant differences: * p < 0.0001 and ** p = 0.01 (n = 6–10 cells). C. HeLa cells expressing IR-B-GFP were labeled in vivo with 50 nM BAC-Ins and 1 nM QD655 at 15°C and further either directly fixed in 3.7% PFA or treated with acid solution (0.5 M NaCl, 0.1 M Na-glycine pH 3.0) for 5 min and washed before fixation. The two bottom panels show similar experiments but the cells were incubated at 37°C for 150 min before acid treatment and/or fixation. In all the cases imaging was performed by confocal microscopy (Zeiss LSM 510 Meta). Scale bars: 10 μm. D. HeLa cells expressing IR-B were labeled with 50 nM BAC-Ins and 1 nM QD655 at room temperature. Samples were incubated or not at 37°C and then fixed in 3.7% PFA. Immunofluorescence was performed with antibodies against early endosomes (EEA1) or CD63 (Lamp 1). Secondary antibodies were conjugated with Alexa fluor 555. Imaging was performed by confocal microscopy (Olympus Fluoview FV 1000). Scale bars: 10 μm.