Fluorogold prolong antifade mounting medium

Frozen spleen tissue was embedded in OCT (Tissue-Tek, The Netherlands), and 10 μm sections were cut with the cryostat and mounted on glass slides. Slides were stored for 24 h at −80°C and washed in 1× PBS, followed by a 10-min fixation step of the sections in 4% PFA. Target retrieval was performed in sodium citrate buffer pH 8.5, followed by blocking of the sections in blocking buffer (3% BSA, 0.3% Triton X-100; PBS) for 2 h at RT. The primary antibodies (for a complete overview of antibodies used, see Supplementary Table 2) were diluted in blocking buffer and incubated for 48 h at 4°C, followed by 3 × 30 min washes in PBS at RT. The secondary antibodies were diluted in blocking buffer and incubated for 2 h at RT, followed by 3 × 30 min washes in PBS, and the sections were mounted using Fluorogold prolong antifade mounting medium (Invitrogen, Germany).

Free-floating sections were blocked for 2 h at RT in 3% BSA and 0.3% Triton in Phosphate-buffered saline (PBS) and incubated with primary antibodies [rabbit anti-TMEM119; 1:100 (Abcam), mouse anti-CD11c; 1:100 (Abcam), rat anti-CD169; 1:200 (BioLegend) rabbit anti-CD3; 1:100 (Abcam), mouse anti-CD45; 1:500 (BD biosciences), mouse anti-CD68; 1:500 (Abcam), mouse anti-CD317; 1:100 (R&D Systems), mouse anti-CS-56; 1:200 (Abcam), rat anti-GFAP; 1:200 (Thermo Fisher Scientific)], mouse anti-NeuN; 1:100 (Millipore), for 48 h at 4°C. Sections were washed for 3 × 30 min in PBS and incubated for 2 h at RT with secondary antibodies [Donkey anti-mouse 568, 1:500 (Invitrogen); Donkey anti-rat 488, 1:500 (Invitrogen); Donkey anti-rabbit 568, 1:500 (Invitrogen)]; Donkey anti-mouse 647, 1:1,000 (Invitrogen) together with DAPI (1:1,000). After a second round of washing (3 × 30 min in PBS), sections were mounted with Fluorogold prolong antifade mounting medium (Invitrogen). The list of material used has been summarized in Supplementary Table 2.