Ultrastructural identification of NgCAM-containing somatic endosomes. (A) Hippocampal neurons were infected with AdNgCAM for 24 h and subsequently fixed. NgCAM was detected with anti-NgCAM antibodies and 15 nm gold–protein A. NgCAM is found on the plasma membrane, in numerous MVBs (asterisks), and in small profiles of unknown identity (arrowheads). (B–E) NgCAM-expressing neurons were fed with anti-NgCAM antibodies and 3 nm gold-BSA for 15–60 min and then washed and fixed. Endocytosed NgCAM (15 nm gold) is found in MVBs together with varying amounts of 3 nm gold BSA. The labeled MVB in B contains many 3-nm gold particles (appearing as dark, grainy material) as well as large 15-nm gold particles (arrows). A larger magnification of the boxed region is shown in the inset below to highlight the two different sizes of particles. The MVB in C has few 3-nm gold particles, whereas the one in D has virtually none. (E) Occasionally, NgCAM is found in tubules that are devoid of cointernalized BSA-gold and likely represent RE. Bars, 150 nm.