Luxol Fast Blue MBS

Cryostat sections prepared from 4% paraformaldehyde-perfused control or MOG35-55-induced EAE female mice were subjected to heat-induced epitope retrieval (HIER) and incubated overnight at 4°C with anti-e1 serum and rabbit anti-Cter Kir4.1_356-375 antibody, revealed by AF594-coupled and AF488-coupled secondary antibodies, respectively. Sections were stained with DAPI and coverslipped with anti-fading mounting medium, and pictures were taken at fixed fluorescence exposure. Peptide-N-glycosidase F (PNGase F, New England BioLabs) was used to evaluate the effect of N-linked glycosylation on the anti-e1 reactivities. For this, HIER-treated sections were incubated with 5 U/µl of PNGase F in 10 mM PBS, 10 mM EDTA at pH 7.6 at 37°C overnight, before being processed for immunohistofluorescence.
For fresh-frozen human tissues, 12µm-thick cryostat sections enriched in subcortical WM (Supplementary Table 3) were prepared from selected blocks containing inflamed subcortical WM or NAWM,^{35 (link)} defined from CD68 and Luxol Fast Blue stainings (Supplementary Table 4). Acetone-fixed sections were processed for Kir4.1 immunostaining as described above, and incubated with 0.1% Black Soudan to stain the white matter and hide lipofuscin-driven autofluorescence before covering with anti-fade mounting medium. For quantification, three fields at ×40 objective of subcortical WM per sample were acquired at fixed fluorescence exposure time, and the average level of Kir4.1 immunofluorescence was measured with ImageJ software. Sections from four human renal fresh-frozen biopsies were also used for Kir4.1 immunofluorescence: control cortical pre-implant biopsies from two donors and cortical kidney biopsies with chronic inflammation from two patients with interstitial fibrosis/tubular atrophy.

An autopsy restricted to the brain (including a small portion of cervical spinal cord) was performed on subject BI. The brain was extensively sampled according to the UCLA dementia protocol including representative sections from the frontal, temporal, parietal and occipital cortices, hippocampus, entorhinal cortex, amygdala, basal ganglia, brainstem and cerebellum. Six-micrometre sections were cut from formalin-fixed paraffin-embedded tissue and were stained with haematoxylin and eosin. Several blocks were also stained with Luxol fast blue. Immunohistochemistry was implemented with antibodies to β-amyloid 1–42 (1:150, EMD Millipore, rabbit polyclonal, AB5078P), β-amyloid 1–40 (1:400, EMD Millipore, rabbit polyclonal, AB5074P), phospho-tau (1:200, Thermo Fisher, mouse monoclonal, AT8) and alpha-synuclein (1:450, EMD Millipore, rabbit polyclonal, AB5038). Incubation with primary antibodies was followed by either horse anti-mouse or horse anti-rabbit secondary antibody conjugated to horseradish peroxidase (MP7402 & MP7401; Vector Laboratories, Burlingame, CA, USA). Visualization of antibody reactivity was achieved with N′N-diaminobenzidine as chromogen (no. SK-4100; Vector Laboratories) and then counterstained with haematoxylin. Neuropathologic substrates of dementia were assessed using standard diagnostic criteria.^{19 (link)} The presence of cerebrovascular disease was also evaluated, including cerebral amyloid angiopathy (CAA) graded according to the Vonsattel criteria.^{20 (link)}All three participants with the F388S mutation, 5/8 of the A431E mutation carriers and 2/3 carriers of PSEN1 mutations not causing SP were male. We therefore were unable to assess effects of gender.

Brain donations were processed using published protocols.^{71 (link),72 (link)} Whole brains were received fresh, on wet ice, and gross pathology was evaluated. Once hemi-sected, one hemisphere was fixed in periodate-lysine-paraformaldehyde and stored at 4°C and the other hemisphere was sectioned coronally and flash-frozen using dry ice. Blocks of tissue from the fixed hemisphere were dissected, embedded in paraffin and cut at 10 µm for immunohistochemistry. Using previously described methods, tissue was stained with Luxol fast blue, haematoxylin and eosin, Bielschowsky silver, and with antibodies for p-tau (Ser202, Thr205), β-amyloid, α-synuclein and phosphorylated transactive response DNA-binding protein of 43 kDa. Neuropathologists blinded to the donors’ clinical and athletic histories used established criteria to diagnose and stage neurodegenerative diseases.^{73–79 (link)} National Institute on Aging-Reagan was followed for the diagnosis of Alzheimer’s disease.^{74 (link)} Neuropathological diagnosis of CTE was made using criteria defined by the NINDS-NIBIB Consensus Conference.^{12 (link),13 (link)} The Understanding Neurologic Injury and Traumatic Encephalopathy study has followed recommendations from the second NINDS-NIBIB Consensus Conference for the neuropathological diagnosis of CTE,^{13 (link)} which requires tau inclusions in neurons with variable astrocytic involvement. It states, ‘…the perivascular p-tau aggregates should include neurofibrillary tangles, with or without astrocytes…’^{13 (link)} CTE stage was also designated using the McKee staging scheme, classified as low (stage I/II) and high (stage III/IV).^{13 (link),52 (link),80 (link)}A modified version of the ischaemic injury scale^{81 (link)} was used as a global indicator of cerebrovascular disease and based on the presence of hippocampal sclerosis, infarcts, microinfarcts, microbleeds, laminar necrosis, arteriolosclerosis, atherosclerosis, cerebral amyloid angiopathy and white matter rarefaction. Unlike the original ischaemic injury scale, the cribriform state was not included because it was not rated in more remote cases from our brain bank. Arteriolosclerosis, atherosclerosis, cerebral amyloid angiopathy and white matter rarefaction were rated on a semi-quantitative scale (0 = none, 3 = severe), whereas the remaining pathologies were rated as absent/present. Methods for the detailed assessments of the individual-level cerebrovascular and white matter pathologies have been described elsewhere.^{54 (link)} The modified ischaemic injury scale is a summary composite of all the pathologies with a possible range of 0–17.
Neuropathological ratings and diagnoses were conducted by study co-authors (B.R.H., A.C.M. and T.D.S.). They have previously been shown to have very good inter-rater reliability on semi-quantitative rating scales of neuropathology, namely scales of CTE and cerebral amyloid angiopathy severity.^{34 (link),80 (link)} While inter-rater reliability across other various individual pathologies is not known, the primary outcome and focus of this study is the quantitative measures of myelin loss (i.e. MAG and PLP) as opposed to the semi-quantitative rating scales.