For peripheral blood analysis, tailvein blood was collected and RBCs were lysed with ammonium chloride buffer prior to staining for flow cytometry.
Samples were then washed three times for 15 min each in wash buffer (2 mM MgCl2 and 0.1% Nonidet P40 in 0.1 M phosphate buffer) and stained for up to 12 h in a solution consisting of 1 mg/ml X-gal (Melford), 5 mM K3Fe CN 6 and 5 mM K4Fe CN 6 in wash buffer.
Then, the cells were rinsed and resuspended in FACS buffer and stained for 25 min with anti-CD45 (#30F-11; Biolegend), anti-CD11b (M1/70; BioLegend), anti-MHC-class II (#M5/114.15.2, eBioscience, San Diego, CA), anti-F4/80 (#Biolegendegend), CD11c (#N418; Biolegend) and anti-siglecF (#E50-2440, BD Pharmingen, San Diego, CA) for macrophages, dendritic cells and eosinophils.
After incubation, the cells were washed in FACS buffer and stained for flow cytometry using rat-anti-mouse CD4 PerCP and rat-anti-mouse CD8 FITC on ice.
Briefly, mouse brain sections were washed with Tris buffer and stained for 10 min with a solution of 0.5% Thioflavin S in 50% ethanol, followed by 50% ethanol and Tris buffer, then dried and covered using Vectashield (Vector Labs, CA).
After the incubation time, cells were washed with FACS buffer, and stained for 20 min at 4°C with PE anti-mouse CD4, PerCP anti-mouse CD8, APC anti-mouse CD45.2 (BD Biosciences Pharmingen) or the isotype controls.
At the end of the incubation, cells washed three times with FACS buffer and stained for 20 min at 4°C in the dark with anti CD11c-PE (1∶400) in the presence of Fc block.
For FACS analysis of reticulocytes, 3 5 μL of heparinized blood was washed in staining buffer and stained for CD71.
Cells were washed once with FACScan buffer then stained for CD8a and IFN-γ as described above.
