Whole brains were harvested from experimental animals at the time points indicated. For paraffin-embedded sections, brains were fixed in formalin acid alcohol (4% formaldehyde and 5% glacial acetic acid in 70% ethanol), paraffinized in a Tissue-Tek VIP, and embedded with a Tissue-Tek TEC. For frozen sections, tissues were fixed in 4% paraformaldehyde in PBS, cryoprotected with 10 and 20% sucrose in PBS, and embedded in optimal cutting temperature compound (4583, Tissue-Tek) or Neg-50 (6502, Thermo Fisher Scientific). Sections were cut to a 10-μm thickness on a Leica microtome or cryostat.

A ZEISS Imager.M2 was used to obtain all images. For bright-field imaging, brain sections were deparaffinized and rehydrated through xylene and ethanol washes, stained with H&E, dehydrated in ethanol and xylenes, and then coverslipped and preserved with dibutylphthalate polystyrene xylene mounting media. Electron microscopy was performed at The Scripps Research Institute Core Microscopy Facility with a Philips CM100 TEM and Hitachi S4800 SEM, with tissue processing, sectioning, and imaging performed with the core staff.

For immunohistochemical analyses, deparaffinized or frozen sections were washed in tris-buffered saline (TBS) (pH 7.4), blocked in TBST (0.1% Triton X-100 in TBS) with 5% normal goat serum (NGS), and incubated with primary antibody in TBST with 1% NGS overnight at the dilutions listed below. After washing in TBST, sections were incubated with secondary antibody diluted 1:1000 in TBST for 1 hour and then washed sequentially in TBST, TBST with 1:10,000 4′,6-diamidino-2-phenylindole nuclear counterstain, and TBS. Slides were coverslipped with VECTASHIELD (H-1000, Vector Laboratories) and kept at 4°C until imaging. Antibodies used were S100A/B (1:300; NB200-538, Novus Biologicals) on paraffinized tissue, AcTub (1:250; T6793, Sigma-Aldrich) on paraffinized tissue, cleaved Cas3 (1:200; #9664, Cell Signaling Technology) on frozen tissue, and Iba1 (1:250; 019-19741, Wako) on frozen tissue. ISEL+ was performed on paraffinized tissue with the 2.5 brown detection kit (322310, Advanced Cell Diagnostics) and horseradish peroxidase labeling (Vector ImmPRESS, MP-7401) as per the manufacturer’s instructions.

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