Immunohistochemistry of human post mortem MS tissue (see Supplemental Data Table 3) was completed on 20 μm thick snap-frozen sections, fixed with ice-cold methanol. Blocking steps included peroxidase blocking with H2O2, avidin and biotin blocking (Vector), and normal serum blocking with 10% serum of species in which secondary antibodies were raised, diluted in 1× phosphate-buffered saline (PBS) with 0.01% Triton-X (PBST). Incubations with primary antibodies diluted in PBST were overnight at 4 °C (mouse anti-rat MOG, Millipore MAB5680 1:1000; MHC-II, abcam HLA DR ab80658 1:50; mouse anti-human CD68, AbD Serotec MCA1815 1:200; rat anti-GFAP, Invitrogen MA5-12023 1:500; mouse anti-human S100A10, Invitrogen MA5-15326 1:1000; rabbit anti-human C3D, DAKO A0063 1:1000), and detection was achieved by signal amplification using biotinylated secondary antisera (Vector) followed by avidin–peroxidase (Vectastain ABC; Vector). Diaminobenzidine (DAKO) was used as chromogenic substrate. Negative control sections without primary antibodies were processed in parallel. Sections were counterstained with either hematoxylin, or 4′,6-diamidino-2-phenylindole (DAPI). Double and triple fluorescence staining was performed with fluorochromes tagged to streptavidin and secondary antibodies (Alexa Fluor 488, 594, and 647, Invitrogen).
