A 38-year-old Iranian man presented with a nine-month history of cerebellar ataxia and dysarthria, weight loss, and vomiting. Brain MRI showed cerebellar atrophy. CSF analysis showed CSF-restricted oligoclonal bands and 5 cells/mul. No further clinical data were available from this patient retrospectively.
IHC was performed on cryosections of adult rhesus monkey, rat, and mouse cerebellum tissue (Euroimmun, Luebeck, Germany) as previously described. Briefly, sections were pretreated with formalin and 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) in PBS, blocked with 10% goat serum and then incubated with patient serum for 1 h. Binding of human immunoglobulin (Ig)G, IgA, and IgM to CNS tissue was detected by using polyclonal goat anti-human IgG antibodies conjugated to fluorescein isothiocyanate (FITC) (Euroimmun) or Alexa Fluor  (AF) 568 (Invitrogen, Karlsruhe, Germany), and polyclonal goat anti-human IgM and anti-human IgA antibodies conjugated to FITC (Euroimmun), respectively. Sections were then mounted using glycerol standard immunofluorescence mounting medium containing 4',6-diamidino-2-phenylindole (DAPI) (1:1000) (Euroimmun) or ProLong Gold antifade reagent (Invitrogen). Slides were analyzed on a Nikon 90i upright fluorescence microscope and a Nikon A1 confocal microscope (Nikon Imaging Center, University of Heidelberg, Heidelberg, Germany).
To evaluate IgG subclasses, serum and CSF samples were tested by IHC on mouse cerebellum sections as described above, with the following modifications: sections were blocked with 10% donkey serum; nonconjugated sheep anti-human IgG antibodies specific for IgG subclasses 1 to 4 (Binding site, Germany) were substituted for the FITC-labeled goat anti-human IgG antibody; and AF568-labeled donkey anti-sheep IgG (Invitrogen; absorbed against human IgG) was used to detect the subclass-specific antibodies.
To confirm ARHGAP26 specificity, sera were preadsorbed overnight with human full-length ARHGAP26 (Biozol, Eching, Germany) and supernatants were tested by IHC as described above.
Protran BA79 nitrocellulose membranes (0.1 mum) (Whatman, Fisher Scientific, Schwerte, Germany) were spotted with a 0.14 mug/mul solution of human full-length ARHGAP26 (10 mul/spot; Biozol). After drying, membranes were blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline (TBS) for 1 h at room temperature (RT), washed three times in TBS with 0.05% Tween (TBS-T), and then incubated with a 1:20 dilution of the patient's serum in 0.1% BSA/TBS-T for 1 h at RT. A donkey anti-human IgG antibody labeled with IRdye 700DX (Rockland, Gilbertsville, PA, USA) was used to detect bound IgG. Stripes were finally washed in TBS and analyzed using an Odyssey  fluorescence scanner (Licor, Lincoln, NE, USA) and Odyssey  2.0.40 application software (Licor).
All samples were tested for anti-Hu, -Yo, -Ri, -CV2/CMRP5, -Ma/Ta, -Tr, -amphiphysin, -aquaporin-4, -GAD, -MAG, and -myelin antibodies by IHC on mouse, rat, and monkey brain and peripheral nerve tissue sections (Euroimmun, Luebeck, Germany), for anti-Hu, -Yo, -Ri, -CV2/CMRP5, -Ma/Ta, and -amphiphysin antibodies by using a commercially available line blot assay (Euroimmun, Germany), and for anti-NMDA-type glutamate receptor, anti-AMPA-type glutamate receptor, anti-GABA-B receptor, anti-CASPR2, anti-LGI1, anti-AQP4, and anti-glycine receptor antibodies using a panel of recombinant cell-based assays (Euroimmun, Germany).