Cerebellar Immunohistochemistry Profiling

For immunohistochemistry staining, cerebellar slices were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) during 24 h at 4°C after ex vivo incubation with the different treatments. Immunohistochemistry was performed as in Arenas et al. (25 (link)). Samples were embedded in paraffin and 5 μm sections were cut and mounted onto coated glass slides. Sections were hydrated, incubated with 3% H₂O₂ for 15 min to block endogenous peroxidases, and with 6% normal goat serum for 1 h. Sections were then incubated with primary antibodies against Iba1 (1:300, Wako), GFAP (1:400; Sigma), TNFα (1:300, Abcam) and Glutaminase 1 (1:100, Novus Biologicals) overnight at 4°C. Next, sections were incubated with secondary biotinylated antibodies goat anti-rabbit and goat anti-mouse (1:200, Vector Laboratories) for 1 h at room temperature. Then, sections were incubated with ABC complex (Vector Laboratories) during 30 min, followed by diaminobenzidine (DAB substrate kit, Abcam) until desired color was acquired (for a maximum of 10 min). Finally, sections were counterstained with hematoxylin (Dako). Sections were scanned with an Aperio Versa system (Leica Biosystems).