IRF8+ nuclei isolation and sorting

IRF8⁺ nuclei were isolated and sorted as described previously [11]. Briefly, frozen tissue from MS donors, NAWM tissue (n = 7) and tissue containing MS lesions (n = 5), matched for age, was provided by the NBB. For each tissue block, the first and last section were double stained for HLA-DR/PLP to determine microglia activation and myelin integrity. MS lesions were characterized as previously described by Luchetti and colleagues [5].

From each tissue block, 10–12 sections of 50 μm thickness were cut and homogenized in 1 ml homogenization buffer (1 μm DTT (Thermo Fischer Scientific), 1x protease inhibitor (Roche), 80 U/ml RNAseIN (Promega, Madison, WI, USA) and 1% Triton X-100) with nuclei isolation medium #1 (NIM #1; 250 mM sucrose, 25 mM KCL, 5 mM MgCl₂, 10 mM Tris buffer pH 8 diluted in nuclease free water) filtered through a 30-μm cell strainer. The amount of nuclei was counted using a hemocytometer (Optic Labor) and nuclei were incubated with Hoechst (#H3570, 1:1000; Invitrogen) and IRF8 antibody (#566373, PE-labeled, 1:50, clone U31–644; BD Biosciences, San Diego, CA, USA) in staining buffer (0.5% RNAse free BSA, 1% normal human serum and 0.2 U/μl RNAseIn in RNAse-free PBS, pH 7.4) for 1 h at 4 °C. Isotype control antibody IgG-PE (#12–4714-42, clone P3.6.8.1, 1:25; Invitrogen) was used to determine background staining.

Stained nuclei were sorted using a Sony SH800S cell sorter (Sony Biotechnology, San Jose, CA, USA). The Hoechst and IRF8 double positive nuclei fractions was collected and lysed in RNA lysis buffer (RNeasy Isolation mini kit; Qiagen, Hilden, Germany).