Figure 3

Demonstration of cytoplasmic and nuclear accumulation of antibody in cultures incubated with anti-Hu or anti-Ri antibody. The intracellular distribution of anti-Hu or anti-Ri antibody was assessed after cultures were incubated with either anti-Hu or anti-Ri antibody, for 6, 24 or 72 hours, using serial confocal images through individual neurons. Anti-Hu and anti-Ri immunoglobulin (Ig)G could be detected in both cytoplasm and nuclei of neurons as early as after 6 hours of incubation, with progressive accumulation of antibody over time. Neurons incorporating IgG excluded SYTOX dyes, indicating that antibody uptake had occurred in living cells. (A-C) Serial images of neurons within hippocampal slice cultures incubated with anti-Hu antibody for 24 hours. (D-F) Serial images of neurons within cerebellar slice cultures incubated with anti-Hu antibody for 24 hours. (G-I) Images of neurons in slice cultures of hippocampus following 72 hours of incubation with anti-Ri antibody. (J-L) Images of neurons in slice cultures of cerebellum following 72 hours of incubation with anti-Ri antibody. Arrows identify the same individual neurons at different levels in each of the three images. Magnification bar indicates 20 μm.