Mca839g

Histochemistry and immunohistochemistry used in this study have been previously described [32 (link)]. Briefly, double-hemispheric tissue sections were cut with a tetrander microtome at a thickness of 10 μm and mounted on large glass slides. For basic classification of pathology and demyelination, sections were stained with Hematoxylin and Eosin (H&E) and Luxol fast blue (LFB) myelin staining (Division Chroma). PLP delivers a good intracortical myelin contrast which is better than that observed with LFB [39 (link)]. Therefore, PLP intensities were used to evaluate and analyze ROIs within the cortex. Iron was detected with 3,3′-diaminobenzidine-tetrahydrochloride (DAB) enhanced Turnbull blue staining [30 (link)]. Immunohistochemical staining for myelin (proteolipid protein, PLP) was performed with a primary anti-human PLP monoclonal mouse antibody (# MCA839G, AbD Serotec, Oxford, UK). Sections were steamed for 60 minutes in EDTA buffer pH 8.5. The primary antibody was diluted 1:1000 in 10% fetal calf serum in Dako wash buffer (FCS/DAKO) and applied at 4°C overnight. The next day, an appropriate biotinylated secondary anti-mouse antibody was applied. After incubation of the slides with avidin-conjugated horseradish peroxidase, the stainings were developed with DAB. Sections were coverslipped with Eukitt.

The visualization, definition, and calculation of proportion of lesion subtypes is described in Luchetti et al 2018 41. In short, double immunostaining was performed on sections from all formalin fixed and paraffin embedded tissues blocks that were dissected from a donor to visualize proteolipid protein (PLP) (MCA839G, AbD Serotec, Oxford, UK, with DAB) and human leukocyte antigen (HLA‐DR‐DQ, referred to as HLA) (M0775, CR3/43, DAKO, Denmark, with DAB‐nickel), as previously described 41. On average, 19.4 ± 12.4 (mean ± SD) tissue blocks were characterized per case. The different lesion characteristics are presented in Figure 1.

IHC was performed on formalin-fixed paraffin-embedded 5 μm–thick sections with EnVisionTM FLEX IHC system (DAKO). Steam antigen retrieval with citric acid buffer (pH 6.0, DAKO) was performed for MAG, MOG, and C9neo staining. Primary antibodies were incubated at 4°C overnight to identify MAG (1:1,000, ab89780, Abcam), MOG (1:1,000, ab109746, Abcam), PLP (1:500, MCA839G, Serotec), C9neo (1:200, monoclonal B7 and polyclonal, from Paul Morgan, Cardiff, United Kingdom), and CD68 (1:100, M0814, DAKO).