Rca 1

Tyrode-HEPES buffer was purchased from BioSolution (Suwon, Republic of Korea). Phosphate buffered saline (1×) without calcium and magnesium (#17-516F) was purchased from Lonza (Basel, Switzerland). Acid-citrate-dextrose (ACD) solution (#C3821), prostaglandin E1 (PGE1; #P5515), neuraminidase (sialidase) from Clostridium perfringens (#11585886001), and OS (#SML1606) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Erythrina crista-galli lectin (#FL-1141-5), SNA (#FL-1301-2), and RCA-I (#FL-1081-5) were purchased from Vector Laboratories (Burlingame, CA, USA). Maackia amurensis lectin II (#21511103-1) antibody was purchased from bioWORLD (Dublin, OH, USA). Anti-CD41 (#11-0411-82) and anti-CD62P (#17-0626-82) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Anti-fibrinogen antibody (ab34269) was purchased from Abcam (Cambridge, UK). Monoclonal antibodies against JAK2 (#3230), phospho-JAK2 (#3776), STAT3 (#12640), and phospho-STAT3 (#9145) were purchased from Cell Signaling Technology (Danvers, MA, USA). Mouse Thrombopoietin Quantikine ELISA Kit (#MTP00) was purchased from R&D Systems (Minneapolis, MN, USA). Mouse TNF ELISA Set (#555268) and Mouse IL-6 ELISA Set (#555240) were purchased from BD Biosciences (San Jose, CA, USA).

All materials were purchased from Sigma-Aldrich Merck (Gillingham, UK) unless otherwise stated. Vectors pCR8/GW/TOPO and pSecTag/FRT/V5-His-TOPO, and the Gateway (GW) vector conversion system were obtained from Invitrogen (Carlsbad, CA, USA). LR Clonase II and the Flp-In-CHO cell line were also from Invitrogen (Carlsbad, CA, USA). FITC-labelled lectins SNA-I and MAA were from EY Laboratories (San Mateo, CA, USA) and biotinylated SNA-I, RCA-I, and MAA lectins were from Vector Laboratories (Peterborough, UK). Oligonucleotide primer synthesis and DNA sequencing was carried out by Eurofins. Proof-reading Phusion High-Fidelity DNA Polymerase (New England Biolabs, Ipswich, MA, USA) was used for PCR amplification of cDNA during construction and standard Taq polymerases (Promega, Madison, WI, USA) for screening. PNGase F was from New England Biolabs (Ipswich, MA, USA).

Total PcH carbohydrate content was determined by the phenol sulfuric acid method (Dubois et al., 1956). Biotinylated lectins were used for the analysis of PcH glycosylation pattern, namely PVL, BSL 1, JAC, SBA, Con A, WGA, DBA, UEA 1, PNA, RCA I, ECL, PSA and LCA (Vector Labs, Burlingame, CA, USA). Dots containing 5 μg of PcH were adsorbed onto nitrocellulose strips (Hybond, GE Healthcare, Uppsala, Sweden) and incubated for 1.5 h at 37°C in a humid environment. After washing with PBS-Tween 0.1%, the strips were then blocked overnight at 4°C with 3% (w/v) oxidized BSA in PBS. Then, each strip was incubated 1.5 h at 37°C with a different lectin at limiting concentrations to avoid nonspecific binding. After five washes in PBS-Tween 0.1% binding was detected using a horseradish peroxidase–streptavidin conjugate (Vector Labs) and visualized by chemiluminescence using the Immobilon Western Chemiluminescent HRP Substrate (Sigma-Aldrich) in a ChemiDoc MP Imaging System (Bio-Rad Laboratories, Inc.) and analyzed with ImageJ software (NIH).

Biotinylated Riccinus communis agglutinin (RCA1; Vector), diluted 1:1000 (2 μg/ml), was incubated with the CNS samples overnight to identify the microglia and macrophages. The antigen was retrieved in citrate buffer (pH 6.0) after irradiation of the samples for 6 min at full power in a domestic microwave.

Tissue processing was performed as previously described [82 (link)]. At week 5.5, a time point of early remyelination, deeply anaesthetized mice were perfused transcardially with 4% paraformaldehyde (PFA, Merck). Brains were removed, postfixed in 4% PFA, and paraffin-embedded. There were 7 μm serial coronal sections cut on a bright rotary microtome (RM2245, Leica) from −0.82 mm bregma to −1.70 mm bregma. A group size of 4 to 6 animals was evaluated immunohistochemically. Histology for Luxol-fast blue periodic acid-Schiff base (LFB-PAS) and immunohistochemistry were performed as previously described [56 (link)]. For immunohistochemistry, paraffin-embedded sections were dewaxed and heat-unmasked in 10 mM citrate buffer (pH 6.0). The following primary antibodies were used: for myelin proteolipid protein (PLP) (mouse monoclonal IgG2a, 1:500, Serotec) and myelin basic protein (MBP) (mouse monoclonal IgG2b, 1:500, Covance), for activated microglia Mac-3 (rat IgG, 1:500, BD Pharmingen) and the lectin ricinus communis agglutinin 1 (RCA-1) (1:1000, biotinylated, Vector Laboratories), for astrocytes glial fibrillary acidic protein (GFAP) (mouse IgG, 1:200, Millipore), for oligodendrocytes Nogo-A (rabbit IgG, 1:750, Millipore) and anti-adenomatus polyposis coli (APC; mouse IgG; 1:200).