Animals and tissues were processed for IHC as described in our previous study (22 (link)). The tissues used for IHC and densitometry analysis in Schartz et al. (22 (link)) were further analyzed for microglia/macrophage cell and morphology counts in the current study. Briefly, animals were anesthetized with beuthanasia (200 mg/kg) and perfused with ice cold 1× PBS followed by 4% paraformaldehyde (PFA). After overnight post-fixation (4%-PFA) and cryoprotection (30% sucrose), brains were frozen in pre-chilled isopentane and stored at −80°C until used. Coronal brain sections (50 µm) were stored in 1×PBS + 0.1% sodium azide at 4°C. Serial sections from rostral to caudal (4–6 sections per brain) were collected at approximately every 500 µm along the dorsoventral axis between the Bregma coordinates −3.00 mm and −5.28 mm. These sections were immunostained with anti-rabbit IBA1 (1:500; Wako Chemicals Cat# 019-19741, RRID: AB_839504) followed by biotinylated goat anti-rabbit secondary antibodies (1:2,000; Vector Laboratories Cat# BA-1000 RRID:AB_2313606), incubated in ABC avidin/biotin complex solution and developed using the DAB Peroxidase (HRP) Substrate Kit, 3,3′-diaminobenzidine (Vector Laboratories). Brain sections were mounted on gelatin-coated slides, Nissl stained, dehydrated in alcohol, de-fatted in Xylene, and coverslipped using Permount mounting media. All chemicals were obtained from Fisher Scientific unless otherwise indicated.
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