Myelin Basic Protein

Stable transfectants (Supplementary Table 2) of the TxB hybrid cell line T2 (MHC-II⁻/DM⁻/DO⁻)40 (link) or Schneider 2 Drosophila melanogaster insect (S2) cells (Life Technologies) were constructed for protein expression and native or recombinant HLA proteins purified as detailed in Supplementary Information. Biotinylated peptides (synthesized by Genscript) include: HA_306–318 (influenza hemagglutinin residue 306–318: PKYVKQNTLKLAT), which binds DR41 (link); EBV_486–500 (Epstein-Barr virus nuclear antigen 1 residue 486–500: RALLARSHVERTTDE), which binds DQ6 (ref. 42 (link)), MBP_86–99 (myelin basic protein residue 86–99: NPVVHFFKNIVTPR), which binds DQ1 (ref. 43 (link)).

The following antibodies were used. The supplier, RRID number, and antibody concentration are provided. Chicken anti-neurofilament [NF, (Merck/Millipore, Burlington, Massachusetts, USA, RRID: AB_177520; 1:2000)], goat anti-choline acetyltransferase [ChAT, (Merck/Millipore, Burlington, MA, USA, RRID: AB_2079751, 1:100)], rabbit anti-myelin basic protein [MBP, (Merck/Millipore, Burlington, MA, USA, RRID: AB_94975, 1:100)] and rabbit ant tyrosine hydroxylase [TH, (Merck/Millipore, Burlington, MA, USA, RRID: AB_390204, 1:250)].

After MR imaging was completed, brains were cryoprotected in 30% sucrose solution for 12 h and frozen on dry ice. Coronal sections (30 µm) were cut and collected on Superfrost Plus (Fisher Scientific, Pittsburg, PA, USA) microscope slides (4 sections) and as free-floating sections stored in cryoprotectant (4 sections). This sequence was repeated every 120 µm. Tissue sections were placed on slides then processed for Cresyl violet (0.1%) staining as previously described [33 (link)]. Free-floating sections were processed for parvalbumin (PV) immunohistochemistry using a modified protocol [7 (link)]. Briefly, sections were removed from cryoprotectant solution and rinsed by 3 washes in PBS. Sections were then treated for 5 min with 3% H₂O₂ followed by non-specific sites blocking using 10% goat serum and 0.2% Triton X for 1.5h. Sections were incubated overnight with a PV antibody (1:200, #NB120−11427, Novus Biologicals, Littleton, CO, USA, RRID:AB_791498) at 4 °C in the blocking buffer. After 3 rinses in PBS, sections were incubated with biotinylated goat anti-rabbit IgG antibody (1:500, #BA-1000, Vector laboratories, Burlingame, CA, USA, RRID:AB_2313606) in PBS with 5% goat serum and 0.01% Triton X for 1 h at room temperature. Following another 3 washes in PBS, sections were incubated in the avidin–biotin–peroxidase complex solution (Vector laboratories) for 30 min. Sections were then incubated in 0.012% 3,3′ diaminobenzidine solution containing 0.01% H₂O₂ to produce a brown reaction product. Sections were mounted on slides and dehydrated prior to being cover-slipped using Permount mounting medium (Fisher Scientific). For the MBP (myelin basic protein) staining, free-floating sections were washed, incubated in blocking buffer (2% goat serum in PBS) for 1.5 h, then incubated overnight with anti-rat MBP antibody (1:250, MAB386, Millipore, Billerica, MA, USA, RRID:AB_94975) in PBS with 0.5% bovine serum albumin. Alexa fluor 488 goat anti-rat IgG secondary antibody (1:1000, A-11006, Life Technologies, Carlsbad, CA, USA, RRID:AB_2534074) was used for fluorescent detection of MBP.
Histological and immunohistochemical stained slides were imaged using a Keyence BZ-X700 microscope (Keyence Corp., Osaka, Japan) capturing the entire section at magnification 10X which was reconstructed using the XY-stitching feature. Total brain and BLA areas were manually delimited using anatomically defined boundaries (PV staining, internal capsule laterally, central nucleus medially and extending up ventrally to ~1.2 mm from the base of the cortical tissues) and data were extracted from each section using BZ-X analyzer software (version 1.2.0.1, RRID:SCR_017375) with the exact same ROI protocol as used for the MRI data. Histological BLA data were obtained by an investigator who did not undertake the MRI analysis.