Figure 4: Monitoring of intracellular trafficking of anti-VEGFR2 Ab by confocal microscopy. After allowing fluorescently labeled anti-VEGFR2 Ab to bind to adherent LSECS (~80% confluent) for 60 min at 4°C, unbound Ab was washed away, and the cells were allowed to internalize surface-bound anti-VEGFR2 at 37°C in the presence of unlabeled VEGF-A and LysoTracker Red (LTR). (a) Shown are representative fluorescence images from each time point (n = 3). Images in each column in the top panel show anti-VEGFR2 antibody (left), LTR (center), and fluorescence overlay (right). In the merge images green color = anti-VEGFR2 antibody, red = LTR, and yellow = their colocalization, highlighting the gradual accumulation of internalized anti-VEGFR2 within acidic compartments (late endosomes/lysosomes). (b) The bottom panel shows live cell imaging of a fusion event.