Microglia Enrichment and Immunophenotyping

Hippocampi were collected and microglia were enriched by using density gradient separation and were prepared as described previously [12 (link), 65 (link)]. The cell suspension was then incubated with Fc receptor blocking antibody CD16/CD32 (1:200, BD Bioscience) and Fixable Viability Dye eFluor® 780 (1:1000, eBioscience) for 10 min at 4 °C. Subsequently, the following antibodies were used: anti-CD11b (1:200, clone M1/70, Biolegend), anti-CD45 (1:200, clone 30-F11, BioLegend), anti-CD11c (1:100, clone N418, Biolengend) and for lineage exclusion by a dump gate anti-CD3 (1:300, clone 17A2, Biolegend), anti-CD19 (1:300, clone 6D5, Biolegend), anti-CD45R (1:300, clone RA3-6B2, BD Bioscience), anti-Ly6C (1:300, clone AL-21, BD Bioscience) and anti-Ly6G (1:300, clone 1A8, BD Bioscience) and incubated for 30 min at 4 °C.