Endocytosed NgCAM leaves somatic endosomes and recycles preferentially to the axonal plasma membrane. (A) After 20 min of anti-NgCAM antibody loading (t = 0), intracellular endocytosed NgCAM can be visualized after acid stripping of surface-bound antibodies in somatodendritic but also axonal (arrows) endosomes. (B) After 90 min of chase at 37°C (t = 90′), somatodendritic endosomes are less prominently labeled, whereas axonal endosomes are still clearly detected (arrows). (A′ and B′) Surface reappearance of endocytosed NgCAM in the same cells was assayed with an Alexa 647–goat anti–mouse antibody after acid stripping and chase. No surface labeling was detectable at t = 0 (A′), demonstrating the efficiency of the acid strip. After 90 min of chase, axonal surface labeling was apparent (B′), demonstrating recycling of NgCAM to the plasma membrane. (C) The disappearance of somatic endosomal NgCAM was plotted as percentage of fluorescence remaining after acid stripping over time (open diamonds). Recycling of Tf from somatic endosomes was plotted as well (circles). Surface reappearance of endocytosed NgCAM was plotted as percentage of the chase end point of 2.5 h (triangles and broken line). The mean of four independent experiments is shown. SEM is indicated for each time point. (D) Representative intensity line scans are shown for dendrites at t = 0 (top left), axons at t = 0 (top right), dendrites at t = 90 min (bottom left), and axons at t = 90 min (bottom right). Brightly staining endosomes correspond to the peaks on the trace. The ratio of axon/dendrite average puncta intensity for t = 0 and t = 90 min is indicated to the right of the traces. n = 4 independent experiments.