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. Author manuscript; available in PMC: 2012 Feb 10.
Published in final edited form as: J Control Release. 2010 Nov 1;149(3):323–331. doi: 10.1016/j.jconrel.2010.10.031

Figure 4. Internalized anti-ICAM/α-Gal nanocarriers traffic to lysosomes and colocalize with Gb3.

Figure 4

(a) TNFα-activated HUVECs were incubated at 37°C for 1 h with Texas-Red dextran to label lysosomes, followed by incubation with FITC-labeled anti-ICAM/α-Gal NCs for 1 hour at 37°C. Cells were washed and fixed, or washed and incubated with cell medium for 2 or 4 additional hours (total incubation time: 1, 3, and 5 h) and then fixed. (b) Cells were incubated for 16 h with DGJ and NBD-Gb3 to visualize accumulation of this green-fluorescent substrate analog in intracellular compartments (red pseudocolor) and then incubated with non-fluorescent anti-ICAM/α-Gal NCs as in a. Cells were then washed, fixed, and permeabilized, and NCs were stained with Texas-Red-labeled goat anti-mouse IgG (green pseudocolor). Samples were analyzed by fluorescence microscopy to determine the percentage of (pseudo) green-labeled NCs which colocalized within (pseudo) red-labeled lysosomes or Gb3-positive compartments (yellow, arrows). Dashed lines mark the cell borders, determined by phase-contrast. Scale bar, 10 μm. Data are mean±SEM (n≥23 cells, duplicated).