Kir4.1 Immunohistochemistry in EAE and MS

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.