After embedding the brain samples in paraffin, coronal sections 5 μm in thickness were mounted on γ-aminopropyl Pifithrin�� trimethoxysilane-coated glass slides (Matsunami, Osaka, Japan). All animal experiments were conducted in accordance with the Standards Relating to the Care and Management of Experimental Animals promulgated by Gifu University, Japan (Allowance No. 08119). For immunohistochemistry, deparaffined brain sections were immersed in 10 mM citrate buffer (1.9 mM citric acid, 8.3 mM trisodium

citrate, pH 6.0) for 5 min at 120°C by using an autoclave for antigen retrieval and then incubated with 3% H2O2 for 10 min to block endogenous peroxidase activity. After blocking with 3% BSA solution in PBS, the sections were incubated with MAb 13–27 specific for RC-HL N protein (19), which had been purified with a R788 solubility dmso MAb Trap kit (GE Healthcare, Little

Chalfont, UK) and then biotinylated with an EZ-Link Sulfo-NHS LC-Biotiniylation kit (Pierce, Rockford, IL, USA) in advance. After 2 hr incubation at room temperature, the sections were colorized by the ABC method using a Vecta stain ABC kit (Vector, Burlingame, CA, USA) and 3, 3′-diaminobenzide tetrahydrochloride as a substrate. Nuclei were counterstained with hematoxylin. Overview pictures were scanned in an Epson GT-X770 scanner (Epson, Suwa, Japan). Microscopic photographs were taken with an Axiovert 200 microscope (Carl Zeiss, Jena, Germany). NA cells grown on an 8-well chamber slide (BD Falcon, Franklin Lakes, NJ, USA) were infected with each virus at a MOI of 2. Mock-infected cells were inoculated with diluent (E-MEM supplemented with 5% FCS) alone. The infected cells were fixed with 3-oxoacyl-(acyl-carrier-protein) reductase 3.7% formaldehyde and permeabilized with 90% methanol

at 48 hpi. Apoptotic cells were detected by a TUNEL assay using a Neuro TACS II kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s protocol. The results of TUNEL assays were examined using a BZ-8000 digital microscope (Keyence, Osaka, Japan). We chose five microscope fields at random and determined the ratio of numbers of TUNEL-positive cells to total cells in the five fields (more than 800 cells in each field). Student’s t-test was applied for statistical analysis and P < 0.05 was considered to be statistically significant. Apoptotic cells in infected mouse brains were detected by TUNEL staining of paraffin-embedded sections described above, using a Neuro TACS II kit (R&D Systems) according to the manufacturer’s protocol. Photographs were taken with an Axiovert 200 microscope (Carl Zeiss). Monolayers of NA cells were inoculated with each virus at an MOI of 2. Mock-infected cells were inoculated with diluent alone. After 2 days, cells were lysed with lysis buffer consisting of 20 mM Tris (pH 8.0), 150 mM NaCl, 20 mM 3-([3-cholamidopropyl] dimethyl-ammonio) propanesulfonic acid, 2 mM EDTA and 0.04 mM p-amidinophenylmethylsulfonyl fluoride. The lysate was clarified by centrifugation at 13 000 ×g for 10 min at 4°C.