Cd11c

Brains were sectioned in the coronal plane at 30 μm on a sliding knife microtome with a −25 °C freezing stage. Sections were stored in cryoprotectant at −20 °C until further processing. Visualization of antibody-bound sections for immunohistochemistry (IHC) was performed using biotinylated secondary antibodies, avidin-biotin complex (Elite), and 3,3-diaminobenzadine (DAB) substrate kit (Vector Laboratories). The following primary antibodies were utilized: mouse MHC class II I-A^b (BD Pharmingen, 1:2000); Iba-1 (Wako, 1:5000); ICAM-1 (AbD Serotec, 1:2000); rat anti-CD3 (AbD Serotec, 1:4000); hamster anti-CD3 (Santa Cruz Biotechnology Inc., 1:2000); CD11c (BD Pharmingen, 1:500); and CD11b (Invitrogen, 1:1000). Biotinylated secondary antibodies utilized included goat anti-Armenian hamster IgG (Jackson Laboratories, 1:1000); goat anti-mouse F(ab′)2 (Jackson Laboratories, 1:2000); goat anti-rabbit IgG (Vector Laboratories, 1:1000); and goat anti-rat IgG (Vector Laboratories, 1:2000). Sections used for MHC II, CD11c, CD3, and Iba-1 analysis were counterstained with Methyl Green (Vector Laboratories) according to the manufacturer’s protocol. For immunofluorescence, primary antibodies were utilized at concentrations three times the IHC level. Secondary antibodies bound to Alexa (Invitrogen) or DyLight (Jackson Laboratory) fluorophores were utilized at a dilution of 1:500.

Spleens were smashed and filtered through 40 μm gauze (BD Biosciences). Single-cell suspensions were then stained for flow cytometry with the following antibodies: CD4 Pacific blue (clone GK1.5), CD25 PE-cy7 (clone PC61), FoxP3 AlexaFluor (clone FJK-16a, eBioscience) were used in combination with a FoxP3 staining kit (eBioscience). T_FH cells were detected with the following antibodies: CD4 (clone RM4-5), B220 (clone RA3-6B2), CXCR5 (clone SPRCL5), PD-1 (clone 29 F.1A12), ICOS (clone 7E.17G9), Bcl6 (clone K112-91), and IL-21 (clone FFA21). Further antibodies: CD3 (clone 145-2C11), CD8α (clone 53-6.7), CD11b (clone M1/70), CD11c (clone N418), CD45 (clone 30-F11), CD138 (clone 281-2), F4/80 (clone BM8), Fas (clone 15A7), Ly6c (clone HK1.4), Ly6G (clone 1A8), MHCII (clone M5/114.15.2), TACI (clone ebio8F10-3), and Siglec F (clone ES22-10D8), PNA-biotin (Vector), Streptavidin-PE/CF594 (BD), NKp46 (clone 29A1.4), CD49 (clone DX5), CD16 (clone 93), IL-4 (clone 11B11), IL-17 (clone TC11-18H10.1), IL-9 (clone RM9A4). T_FH:B cell doublet gating strategy is adopted from previously published article^{69 (link)} and is shown in Supplementary Fig. 3f.