Rpn1231v

Proteins were separated by SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked in TBS containing 5 mg/mL non-fat milk and 1% Tween 20 for 1 hour at room temperature. Blots were probed for FLAG (1:5000; F1804; Sigma-Aldrich), β-tubulin (1:5000; E7; DSHB at the University of Iowa), H3K27ac (1:1000; 07-360; EMD Millipore) and Streptavidin conjugated to HRP (1:5000; RPN1231V; GE Healthcare). Detection on film was performed by chemiluminescence using the LumiGLO reagent (Cell Signaling Technology; #7003; 1:20).

Brains were removed from deeply anesthetized and exsanguinated mice, frozen in liquid nitrogen and sectioned in a cryostat. Parallel brain sections (20 μm thick) were fixed 45 min in 4% paraformaldehyde for CD4 immunostaining or 4 min in 4% phosphate buffered formaline, pH 7.4, followed by 2 min in 50%, 100%, and 50% acetone, respectively, and finally air dried for CD8a immunostaining. Sections were then rinsed in 0.5 % triton X-100 diluted in 0.05M trizma base buffered saline (TBS+T), pH 7.4, and incubated with 10% fetal calf serum (FBS) in TBS+T before incubation overnight at 4°C with purified rat-anti-mouse CD4 IgG2a (2.5 μg/mL) (553647, BD Pharmingen) or purified rat-anti-mouse CD8a IgGa (7 μg/mL) (MCA1108GT, AbDSerotec) diluted in 10% FCS in TBS+T. Parallel sections were incubated with rat IgG2a isotype control (2.5–7 μg/mL) (BD Pharmingen). After a rinse in TBS+T, endogenous peroxidase activity was blocked with 1.9% H₂O₂ in TBS, and sections were rinsed in TBS+T before and after incubation with biotinylated goat anti-rat IgG antibody (5 μg/mL) (BA9400, Vector Laboratories Inc) and HRP-conjugated streptavidin (1:300) (RPN1231V, GE Healthcare), respectively, diluted in 10% FCS in TBS+T. After a final rinse in TBS+T, the colour signal was developed in 0.05% 3.30-diaminobenzidine tetrahydrochloride and 0.003% H₂O₂ in TBS. Finally, sections were coverslipped with Depex.