Figure 4

Uptake of anti-Hu antibodies by hippocampal neurons was followed by neuronal death. Hippocampal slice cultures from rat pups at 23 to 24 days of age were incubated for 72 hours with a 1:400 dilution of either anti-Hu or normal control sera. SYTOX green was added to cultures 2 hours prior to harvesting as a marker of cell death. Cultures were fixed and immunostained with Cy5-conjugated donkey anti-human immunoglobulin (Ig)G (red). Multiple neurons exhibited yellow fluorescence, due to the presence of both anti-Hu IgG (red) and SYTOX green, indicative of cell membrane disruption and death in neurons containing anti-Hu IgG. Images shown are representative of experiments investigating all seven anti-Hu samples studied. IgG uptake was not observed in control cultures incubated with normal human IgG, and only rare neurons stained with SYTOX dyes. Magnification bar = 20 μm.