Type I interferon protects neurons from prions in in vivo models

Ishibashi, Daisuke; Homma, Takujiro; Nakagaki, Takehiro; Fuse, Takayuki; Sano, Kazunori; Satoh, Katsuya; Mori, Tsuyoshi; Atarashi, Ryuichiro; Nishida, Noriyuki

After the mice were sacrificed at the time point, the brain and spleen tissues were fixed in 10% formalin and sectioned into 3 mm slices that were then embedded in paraffin. To evaluate spongiform change, the sections were analysed by haematoxylin and eosin staining as previously described (Ishibashi et al., 2012a; Nakagaki et al., 2013) . As per the immunohistochemistry protocol, after deparaffinization and rehydration as preparation for pre-staining, the sections were boiled with Target Retrieval Solution (Dako, S2369), and treated with 0.3% hydrogen peroxidase in methanol to inactivate endogenous peroxidase, and sequentially incubated with 1% bovine serum albumin in Tris-buffered saline-Tween-20 (TBST) at room temperature for 1 h. The sections were reacted with primary antibodies and envision polymer horseradish peroxidaseconjugated anti-rabbit and anti-mouse immunoglobulin G antibodies (Dako, K4002 and K4000) for 1 h at 37 C, respectively, and visualized using 3,3 0 -diaminobenzidine. To detect gliosis, Iba-1 (WAKO, 019-19741) for microglia and GFAP (DAKO, Z033429) for astrocytes, were applied to the sections as primary antibodies. For PrP Sc staining, autoclaving was performed using the hydrochloric acid protocol described previously (Nakagaki et al., 2013) . In this pathological analysis, brain tissues from the regions of the cortex, hippocampus, thalamus, cerebellum, and Pons were evaluated. For semiquantification in histopathological analysis, the pathological degree of each region in the tissues was scored on a 0 to 5 scale (i.e. non-detectable, a few, mild, moderate, severe, and status spongiosis) as described previously (Ishibashi et al., 2012a) .