Histology and immunohistochemistry

For all in vivo histology experiments, animals were killed via lethal overdose with FatalPlus (Vortech Pharma) followed by transcardial perfusion with saline followed by 4% paraformaldehyde, brains removed, and postfixed for 12 hours. Brains were sectioned coronally at 45 μm thickness on a cryostat (Leica) and mounted onto SuperFrost charged slides (Fisherbrand) for subsequent staining procedures.

Mast cells were visualized using staining with acidic Toluidine Blue (Sigma; 0.5% in 60% ethanol; pH = 2.0; 10 minute stain incubation) as detailed in⁷, and then tissue was cleared with ascending ethanol, defatted with xylenes, and coverslipped using Permount.

Brain sections were rinsed twice with PBS, permeabilized with 0.3% H₂0₂ in 50% methanol, blocked with 5–10% bovine serum albumin or normal goat serum in PBS + 0.4% Triton X, and incubated with primary antisera against the pan-microglia marker, Iba1 (Wako cat#019-19741 1:1000) for 24 hours at 4 °C. Sections were extensively washed, and processed with biotinylated secondary antibodies (Vector), avidin-biotin complex (Vector), and reacted with Nickel-diaminobenzidine in 0.125 M sodium acetate to visualize chromogen for IHC. Stained sections were coverslipped with DPX mounting medium.