Streptavidin fitc

Cells were seeded onto poly-l-lysine-coated glass cover-slides. For binding experiments, cells were incubated with biotinylated nanocages in DMEM 10% FBS for 1 h at 4 °C, washed in PBS, fixed in 4% paraformaldehyde, and incubated for 5 min with NaBH₄. For uptake experiments, cells were incubated with nanocages at 37 °C for different time periods, fixed with 4% paraformaldehyde, and permeabilized for 4 min with Tris/Triton (Tris HCl 0.1 M, Triton 0.1%, pH 7.7). Rabbit polyclonal anti-flotillin-1 antibody (Santa Cruz Biotechnology Inc., Dallas, TX, USA) was used to detect the flotillin-1 protein. Early endosomes were visualized with polyclonal EEA1 antibody (Abcam Inc., Toronto, ON, Canada) and lysosomes with mouse monoclonal anti-LAMP-1 antibody (Abcam Inc., Toronto, ON, Canada). Donkey anti-rabbit IgG and donkey anti-mouse IgG, both Rhodamine Red-X-conjugated AffiniPure (Jackson ImmunoResearch, Cambridgeshire, UK) were used as secondary antibodies [24 (link)]. Biotinylated cages were detected by using streptavidin–FITC (Jackson ImmunoResearch, Cambridgeshire, UK). The nuclei were stained with DAPI (Invitrogen, Carlsbad, CA, USA). Images were obtained with a laser confocal fluorescent microscope Olympus FV1000 at 60× magnification, and the fluorescence signal was evaluated with the IMARIS software. Co-localization events were evaluated as previously described [24 (link)].