Cd19 pecy5

Splenocytes were isolated from spleens for flow cytometric analysis. Antibodies against the following surface markers were provided by: CD3 FITC (eBioscience™), CD4 PE (eBioscience™), CD8a Alexa Fluor 700 (eBioscience™), CD161 APC (eBioscience™) and CD19 PE-Cy5 (eBioscience™). Dead cells were stained by LIVE/DEAD® Fixable Blue Dead Cell Stain Kit (Life Technology). For immunohistochemical brain analyses, the left cerebral hemisphere was dissected and post-fixed in 4% paraformaldehyde in 0.1 M PBS for 2 days. Brains were cryoprotected by incubation in a 30% sucrose/0.1 M PBS solution. Sagittal brain sections were cut on a freezing microtome (Leica) and collected serially. Immunohistochemistry was performed on free-floating microtome-cut Sects. (10 μm in thickness). Sections were incubated with different antibodies: anti-mouse Iba1 (Polyclonal, 1:1000 Wako), anti-mouse CD68 (Clone FA-11, 1:200; BioRad), β-Amyloid (Clone 6E10, 1:1000; BioLegend), β-Amyloid 1–42 (polyclonal, 1:100; Millipore), anti-mouse GFAP (Clone GA-5, 1:100, Novus Biological) anti-human CD3 (Clone: CD3-12, 1:100, abcam) and anti-human Foxp3 (Clone 236A/E7, 1:100, Invitrogen). Appropriate secondary antibodies (Alexa Fluor 488, 594, or 647; Invitrogen) were used followed by incubation with DAPI.

Immune cells were harvested from the airways via bronchoalveolar lavage (BAL). Briefly, mice were cannulated via a small tracheal incision and the lungs were flushed with 1 mL of sterile PBS. The collected BAL fluids were centrifuged at 1,500 rpm for 5 min at 4°C, and total cells were prepared for flow cytometry staining to determine the number and types of cells. Fc-blocked (1 μg/mL; eBiosciences) BALF cells were stained with anti-mouse SiglecF-PE (0.3 μg/mL; BD Pharmingen), CD11c-APC (0.3 μg/mL; eBiosciences), CD11b-PerCP (0.3 μg/mL; BioLegend), CD19-PECy5 (0.8 μg/mL; eBiosciences), Ly6G-PECy7 (0.8 μg/mL; BioLegend), Ly6C-APC-Cy7 (0.8 μg/mL; BD Pharmingen), and TCRβ-Pacific Blue (0.3 μg/mL; BioLegend). All samples were analyzed on a Becton-Dickinson LSR-II/Fortessa flow cytometer (BD Biosciences, San Diego, CA, USA) and analyzed by using FlowJo software (Tree Star Inc.).