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Figure 1 | Journal of Neuroinflammation

Figure 1

From: Neuronal uptake of anti-Hu antibody, but not anti-Ri antibody, leads to cell death in brain slice cultures

Figure 1

Real time uptake of Cy5-conjugated anti-Hu immunoglobulin G in living cultures compared with uptake in parallel cultures examined following fixation. Cultures were incubated with Cy5-conjugated anti-Hu immunoglobulin (Ig)G and were examined in situ for antibody uptake at 6 and 24 hours. Anti-Hu IgG was visible within multiple neuronal populations at 6 hours, with an increase in numbers of antibody-containing neurons and intensity of immunolabeling for intraneuronal IgG by 24 hours. Parallel cultures at both time points were treated with SYTOX dyes and fixed with 2% paraformaldehyde. Uptake of Cy5-conjugated anti-Hu IgG in these cultures was indistinguishable from that observed in slice cultures studied in real time, confirming that IgG uptake observed in cultures fixed following incubation with antibody accurately reflected events occurring prior to fixation. Neurons containing Cy5-conjugated anti-Hu IgG at these time points excluded SYTOX dyes, indicating that antibody uptake had occurred in living cells. Images are representative of real-time studies employing anti-Hu sera from two different patients, carried out in triplicate. Magnification bar indicates 20 μm.

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