Ab150172

Brains were postfixed overnight in 4% PFA before being dehydrated with sucrose solution (10%, 20%, 30%) for 3 days at 4 ℃. Brains were cut into 50-μm-thick sections in the coronal plane. Sections containing DLS and DMS were rinsed with 0.5% Triton X-100 (Solarbio, China) for 5 min, and blocked with 5% BSA (Sangon Biotech, China) for 1 h at room temperature. Then, the sections were incubated with mouse anti-CD31 (ab64543, 1:200, Abcam, UK), rabbit anti-claudin 5 (ab15106, 1:200, Abcam, UK), chicken anti-GFAP (ab4674, 1:2000, Abcam, UK), rabbit anti-Iba1 (ab153696, 1:500, Abcam, UK), rabbit anti-IL17A (A0688, 1:50, Abclonal) and mouse anti-CD4 (67786-1-lg,1:200, Proteintech) antibodies for 48 h at 4 ℃ in the dark. Then, the sections were incubated with Alexa-568 donkey anti-rabbit IgG (ab175470, 1:1000, Abcam, UK), Alexa-488 donkey anti-mouse IgG (ab150105, 1:1000, Abcam, UK), and Alexa-594 goat anti-chicken IgG (ab150172, 1:1000, Abcam, UK) for 2 h at room temperature. Finally, sections were mounted on slides with Fluoroshield™ with DAPI (F6057, Sigma-Aldrich, USA). Images were captured with a Nikon A1R HD25 confocal microscope system (Nikon, Japan). Immunofluorescence quantification was performed as previously described [24 ] and detailed in the Additional file 1.