For P28 PLX-3397-treated animals, brain cell dissociation and staining were performed as described previously15 with minor changes, specifically the addition of cold PBS intravascular perfusion, dissection of cortex rather than whole brain, and the use of a different fluorophore panel. Briefly, cortices from PLX-3397 and control treatments were dissected from anesthetized, cold PBS-perfused mice at P28. Cortices were homogenized in ice cold HBSS supplemented with 15mM HEPES and 0.5% glucose by 5 gentle strokes in a 7 mL glass dounce homogenizer. Dissociated cell suspensions were run through MACS myelin depletion columns, stained with a dead cell marker (LIVE/DEAD, Life Technologies, L23101), and then immunostained using antibodies specific to TMEM119 (custom antibody15, secondary Ab Biolegend 406410), CD45 (eBioscience 25-0451-82), and CD11b (Biolegend 101228). Samples were analyzed on an LSR II (Becton Dickinson), and data processed using Flowjo software (Treestar). Data was collected on an instrument in the Stanford Shared FACS Facility obtained using NIH S10 Shared instrument Grant (S10RR027431-01). Debris, doublets, and dead cells were excluded using fsc/ssc, fsc-h/fsc-w, and green florescence gates, respectively.
