Figure 8

Abolition of anti-Hu cytotoxicity for hippocampal neurons by adsorption of anti-Hu immunoglobulin G with HuD antigen. Rat hippocampal slice cultures were incubated for 72 hours with either native anti-Hu serum (Anti-Hu Sera), the same anti-Hu antibody after passage through a nickel column with bound control vector lacking HuD protein (Sham Adsorbed), or following passage through a nickel column with bound HuD protein (Hu D adsorbed). The upper panels show merged antibody labeled with Cy5 (red) and SYTOX (green). The lower panels demonstrate neurons analyzed only for uptake of SYTOX dyes indicative of cell death. Examples of antibody-positive dead cells are shown with arrows. Adsorption of anti-Hu serum with HuD protein effectively abolished intracellular antibody binding and killing, confirming that cell death was specifically due to interaction of anti-Hu antibody with its target antigen. Similar abolition of antibody uptake and cell killing was also seen in cerebellar cultures (Additional file 3). Images are representative of studies carried out in triplicate. Magnification bar indicates 20 μm.