Permount aqueous mounting media

Immunohistochemistry was performed using standard protocols. Briefly, brains were explanted, fixed in formalin for 24 h and embedded in paraffin. Sections (4 μm) were cut, baked for 1 h at 65°C, de-waxed in 2 successive xylene baths of 5 min and hydrated for 5 min using an alcohol gradient (ethanol 100%, 90%, 70%, 50%, 25%). The slides were incubated in 3% hydrogen peroxide/methanol solution for 10 min. Antigen retrieval was performed using citrate buffer (10 mM sodium citrate, 0.05% Tween™ 20, pH 6) with heating to 95°C in a steamer for 25 min. After cooling down to room temperature, the slides were blocked and incubated overnight at 4°C with a primary antibody against GFAP (1:50; Cell Signaling Technology®, Danvers, MA), IBA1 (1:8,000; Abcam®, Cambridge, MA), NeuN (1:3,500; Abcam) or γ-H2AX (1:1,000; Cell Signaling Technology). The slides were then incubated with ready-to-use IHC detection reagent (100 μl; Cell Signaling Technology) at room temperature for 1 h, rinsed, and incubated with DAB (Cell Signaling Technology) for 3–5 min. Tissues were counterstained or not with Harris modified Hematoxylin (Fisher Scientific™, Waltham, MA) for 30 s, dehydrated via an alcohol gradient (ethanol 25%, 50%, 70%, 90%, 100%) and soaked twice in xylene. A drop of Permount™ aqueous mounting media (Fisher Scientific) was added on the top of the section and covered.

Formalin-fixed, paraffin-embedded brain tissue sections were deparaffinized and hydrated to 95% ethyl alcohol and left in luxol fast blue (Sigma-Aldrich® LLC, MO) solution at 56°C overnight. Slides were then rinsed in the lithium carbonate solution and 70% ethanol (10 s each) followed by counterstaining in Cresyl Violet (Sigma-Aldrich). Tissues were dehydrated in 100% alcohol, soaked in xylene (2 × 5 min) and mounted with Permount aqueous mounting media (Fisher Scientific).
All microscopic images were acquired using a Keyence BZ9000 digital microscope.