For adult mice microglia isolation, brains were quickly removed and washed with prechilled PBS. Then, the brains were cut into small pieces (2 to 3 mm3) and immersed in homogenization buffer [1× PBS containing 2% fetal bovine serum (FBS) and collagenase (1 mg/ml)], followed by incubation for 15 to 20 min at 37°C. A 10-ml syringe plug was used to grind the tissue, and samples were filtered through a 70-μm strainer. The homogenates were then pelleted at 600g for 6 min, resuspended in 2 ml of 37% isotonic Percoll, underlain with 3 ml of 70% isotonic Percoll, covered by 2 ml of 30% isotonic Percoll and 3 ml of 1× PBS, and centrifuged at 2000g for 20 min at 4°C with minimal acceleration and no deceleration. Following Percoll gradient centrifugation, cells at the 37 to 70% interphase were collected and washed with 1× PBS, followed by centrifugation at 600g for 6 min. The pellet was resuspended with staining buffer (1× PBS containing 2% FBS) and incubated with anti-CD11b Alexa Fluor 488 (clone M1/70, 101217, BioLegend) and anti-CD45 phycoerythrin/Cy5 (clone 30-F11, 103110, BioLegend) for 30 min on ice in the dark. Microglia were then purified with a BD Influx and defined as CD11b+ CD45Low single cells and processed for RNA/protein extraction.

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