Ab85809

General reagents were purchased from Sigma-Aldrich and Thermo Fisher Scientific. Human MBP (18.5 kDa isoform) was purchased from Meridian Life Science. The MBP(84–104) (ENPVVHFFKNIVTPRTPPPSQ) based on human MBP sequence (AAH08749, GenBank) and SCR (EFPHIKVTVVTPRNGFPNSPP) peptides, i.e., N-terminally biotinylated and C-terminally amidated peptides, were synthesized by GenScript. Rabbit polyclonal anti-CD20 antibody was obtained from Abcam (ab85809), mouse monoclonal anti-β-actin antibody was obtained from Sigma (A53166), and rabbit anti-degraded (d)MBP antibody, generated against the synthetic peptide encoding guinea pig MBP(69–86), was obtained from EDM-Millipore (AB5864). FluoroMyelin 488 (F34651) was obtained from Molecular Probes. For ELISA, HRP-conjugated goat anti-rat IgM (3020-05) and anti-rat immunoglobulin G (IgG) (112-035-175, Jackson ImmunoResearch), a 3,3′,5,5′-tetramethylbenzidine substrate (TMB/E; Surmodics), and BSA (an IgG and protease-free, 30% solution, United States Biological) were used.

Tissue (e.g., sciatic nerves and spleen) extracts were prepared in TBS and supplemented with 1% Triton X-100, 10% glycerol, 0.1% SDS, 5 mM EDTA, and Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific #11861281). Insoluble material was removed by centrifugation (14,000 × g; 15 min). Extract aliquots (50–150 μg of total protein) were separated by 15% Tris-glycine SDS-gel electrophoresis (Bio-Rad). Separated proteins were transferred onto a PVDF membrane. The membrane was blocked with 5% non-fat milk (Bio-Rad) and incubated for 16–18 h at 4°C with the rabbit polyclonal anti-CD20 antibody (ab85809, Abcam), followed by incubation for 1 h at room temperature with the rabbit-specific HRP-conjugated goat secondary antibody (Cell Signaling; 1:2,000 dilution). Three washes in TBS/T at room temperature were performed after each step. The membranes were re-probed using a β-actin antibody as a loading control (A53166, Sigma). The blots were developed using the SuperSignal West Dura Extended Duration Substrate kit (Thermo Fisher Scientific) and then digitized and quantitated using Image J.