The care and euthanasia of animals were in accordance with University of Colorado IUCAC policy for animal use, which is in agreement with the NIH Guide for the Care and Use of Laboratory Animals.
Myelin-specific MS monoclonal recombinant antibodies (rAbs) used in this study were constructed from expanded CSF plasma blast clones derived from a relapsing-progressive MS patient 6 months after disease onset (MS04-2, for rAb MS04-2#30  or from an isolated optic neuritis patient following their first clinical event who subsequently progressed to clinically-definite MS (ON07-7, for rAb MS07-7#49). NMO monoclonal recombinant anti-AQP4 antibody #53 (NMO#53) was cloned from seropositive NMO patient CSF plasma blast , and the isotype-control human antibody (Iso) was generated from a chronic meningitis patient CSF plasma blast clone . All rAbs were expressed as full-length bivalent human IgG1 antibodies containing a C-terminal Flag epitope using a dual vector transient transfection system and purified with protein A-sepharose (Sigma-Aldrich, St. Louis, MO) as previously described [2, 22]. All rAbs were used at 20 μg/ml in the slice cultures.
Cerebellar slice culture
Sagittal cerebellar slices (300 μm) were prepared from PLP-eGFP mice  at P10 and cultured on MilliCell 0.4 μm membrane inserts (Millipore, Billerica, MA) for 10–14 days in slice media (25% Hank’s balanced salt solution (HBSS), 25% heat-inactivated horse serum, 50% minimum essential media (MEM), 125 mM HEPES, 28 mM D-Glucose, 2 mM L-Glutamine, 10U/ml penicillin/streptomycin, all from Life Technologies, Carlsbad, CA) at 37 °C . Prior to treatment, slices were switched to a serum-free media (Neurobasal medium supplemented with B27, 2 mM L-glutamine, 10U/ml penicillin/streptomycin and 28 mM D-glucose).
Treatment of cerebellar slices
rAbs were applied at 20 μg/ml with or without 10% normal or C5-depleted human serum (Complement Technology, Tyler, TX). Media containing treatment reagents were applied both on top (50 μl) and below (250 μl) the membrane insert. For live binding assays, unfixed slices were incubated with 20 μg/ml rAbs for 4 h at 37 °C. Propidium iodide (PI) (Sigma) was used at 5 μg/ml in the culture medium to label dead cells in organotypic slice cultures. Normal and C5-depleted human serum (Complement Technology, Tyler, TX) were used at 10% (vol/vol) as the source of human complement (HC).
For immunostaining, adult C57bl/6 mice were perfused with 4% paraformaldehyde in phosphate-buffered saline (PBS), and the brain was subsequently removed, post-fixed overnight in 4% paraformaldehyde, and cryoprotected overnight in 20% sucrose at 4 °C. Mouse brain was embedded in optimal cutting temperature (OCT) freezing media, and 6–10 μm cryostat sections collected on Superfrost Plus microscope slides (Fisher Scientific, Pittsburgh, PA). Tissue sections were stored at −80 °C until immunostaining.
After treatment, cerebellar slices were rinsed twice with PBS and fixed in 4% paraformaldehyde in PBS for 20 min at 4 °C. For immunohistochemistry, slices were rinsed in PBS and permeabilized in 1.5 or 10% (myelin proteins) Triton X-100 in PBS for 20 min. Slices were rinsed, blocked with 5% normal donkey serum (NDS) in PBS with 0.3% Triton X-100 for 1 h, and incubated with primary antibodies overnight at room temperature. Following 3 washes in PBS, Alexa Fluor-labeled secondary antibodies (Jackson ImmunoResearch, West Grove, PA) were applied (1:800) overnight at room temperature, washed 3 times in PBS and mounted in Fluoromount G (Southern Biotech, Birmingham, AL). The following primary antibodies were used: rabbit anti-GFAP (Sigma), rabbit anti-Calbindin (Millipore), mouse anti-MAG (Millipore), rabbit anti-MOG (Abcam, Cambridge, United Kingdom), mouse anti-MBP (Covance, Princeton, NJ), chicken anti- Neurofilament-H (Neuromics, Minneapolis, MN), goat anti-Iba1 (Abcam), mouse anti-C3d (a gift from Dr. Joshua Thurman, University of Colorado), rabbit anti-MAC complex (anti C5b-9, Abcam), guinea pig anti- NG2 and rabbit anti-Olig2 (are gifts from Dr. Charles Stiles, Harvard University).
Prior to immunostaining, PFA-fixed mouse cerebellum tissue sections were thawed for 10 min, re-hydrated in PBS for 10 min, and blocked in PBS containing 3% bovine serum albumin (BSA), 2% normal goat serum (NGS), and 0.3% Triton-X100 for 1 h. MS rAbs were applied at a final concentration of 20 μg/mL to mouse tissues for 16 h at 4 °C in PBS containing 3% BSA and 2% NGS. Tissue sections were then washed 3 times for 3 min with PBS. Alexa fluorescent secondary antibody (1:1000) against human IgG (Molecular probes, Life Science Technologies) was applied for 1 h at room temperature in PBS containing 3% BSA and 2% NGS. Tissue was then washed 5 times for 3 min in PBS and mounted using Vectashield (Vector Laboratories, Burlingame, CA, USA) containing DAPI.
Fluorescence images were acquired by Zeiss fluorescence microscope with Axiovision software (Zeiss, Jena, Germany). Confocal images were acquired by Leica SP5 laser scanning microscope (Leica Microsystems GmbH, Wetzlar, Germany). Super-resolution structured illumination microscopy was performed using Nikon’s N-SIM (Nikon, Tokyo, Japan).
Quantification and statistical analysis
PLP-eGFP positive cell bodies in the cerebellar slices were imaged using a Zeiss fluorescence microscope with 20X objective. Images were quantified with ImageJ (National Institutes of Health open source). To quantify the extent of myelination, we calculated the percentage of total neurofilament-H staining that was co-labeled with MBP using a Matlab algorithm (MathWorks, Natick, MA). For each slice, 2–3 images were taken, quantified, and averaged. Slices from 3–4 independent experiments were analyzed. Statistical analyses were performed by unpaired Student’s t test for single comparisons or by two-way ANOVA for grouped comparisons using GraphPad Prism software. Data are expressed as means ± SD of independent experiments (n ≥ 3). Significance is reported for p < 0.05.