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Fig. 1 | Acta Neuropathologica Communications

Fig. 1

From: Induction of aquaporin 4-reactive antibodies in Lewis rats immunized with aquaporin 4 mimotopes

Fig. 1

Characterization of NMO-IgGs used for mimotope search. a-d Immunofluorescence staining of Lewis rat astrocytes and analysis by confocal microscopy. The NMO-IgG preparation IV containing pathogenic AQP4-reactive antibodies recognizing conformational epitopes on the surface of astrocytes (a, red), a commercial AQP4-reactive antibody recognizing intracellular AQP4 epitopes (b, green) and an antibody directed against GFAP (c, blue) were used for stainings. Stainings against surface and intracellular AQP4 epitopes were merged to prove that IV contains AQP4-reactive antibodies reacting with rat AQP4 (d, white). e-f Formation of astrocyte-destructive lesions in experimental NMO. Shown here are spinal cords of Lewis rats injected with myelin basic protein-specific T cells and the NMO-IgG preparations IV (e), III (f) and I (g). Sections were stained with antibodies against AQP4 to show astrocytes (brown) and counterstained with hematoxylin to show nuclei (blue). h For each NMO-IgG preparation used, the phage display peptide library Ph.D.-12 was subjected to three rounds of negative selection on human control-IgG (Subcuvia) to deplete phages binding to “common antibodies”, and of positive selection on NMO-IgG to enrich for phages binding to the AQP4-reactive antibodies contained within the NMO-IgG preparation. At the end of these selections, bound phages were released, amplified, and sequenced for the identification of the mimotopes. i Example of sequencing results for mimotope IV-04. The DNA sequence represents the genomic (+) ssDNA in 5´➔ 3´ direction. Underneath you see the corresponding amino acid sequence (capital letters). Mimotope flanking regions are shown in gray, restriction enzyme recognitions sites in yellow and red, and the mimotope sequence in magenta

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