Fig. 3

Immunofluorescence staining of recombinant flotillin and the neutralization of antibody reaction on tissue. A Immunofluorescence analysis of transfected HEK293 cells. Acetone-fixed recombinant HEK293 cells expressing flotillin-1 (1), flotillin-2 (2), flotillin-1 and flotillin-2 (3) or a mock-transfected control (4) were incubated with patient serum (1–4) or with control serum (1′–4′) (both 1:100). Cell nuclei were counterstained with TO-PRO-3 iodide (blue). Only HEK293-flotillin-1/2 cells reacted with the samples (green). Scale bar: 50 μm. B Neutralization of immunofluorescence reaction on neuronal tissues. Serum was pre-incubated with extracts of HEK293 cells transfected with empty control vector (1–4) or with the plasmid harboring flotillin-1/2 cDNA (1′–4′). The extract containing flotillin-1/2 greatly reduced or abolished the immune reaction of the serum on rat hippocampus (1′) and cerebellum (2′), primate cerebellum (3′), and HEK293-flotillin-1/2 (4′). The control extracts had no effect (1–4). Nuclei were counterstained by incubation with TO-PRO-3 iodide (blue). Scale bar: 50 μm. PS patient serum, CS control serum, sg stratum granulosum, sm stratum moleculare, sp stratum purkinjense