Twenty-four hours after injection with vehicle or LPA into P8 mouse ventricles (P8+1d), animals were perfused with PBS, and brains were harvested. The Miltenyi gentleMACS Octo dissociator was used to create single-cell suspensions from whole brains. Immune cells were then isolated by a discontinuous Percoll (17-0891-02, GE Healthcare) gradient before fixation in 0.1% paraformaldehyde. Cells were incubated with anti-CD16/32 (Fc receptor block; 14-0161, eBioscience) before staining in Brilliant Stain Buffer Plus [566385, BD Biosciences (BD)] with antibody cocktail to identify macrophages and activated microglia (CD45+CD11b+/hiF4/80+Ly6G). Antibodies were purchased from either BD or eBioscience and were as follows: CD45-BV711 (30-F11, BD), CD11b-BV510 (M1/70, BD), F4/80-AF488 (BM8, eBioscience), and Ly6G-PE-Cy7 (1A8, eBioscience). Liquid counting beads (BD) were added immediately before data were obtained on a BD FACSAria Fusion flow cytometer and analyzed using FlowJo v10.4 software (Tree Star Inc.).

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