Normal donkey serum (nds)

The brains of mice infected with 3.0 LD₅₀ of each virus strain via the i.m. route were collected on the day of euthanasia and fixed in 4% paraformaldehyde before being embedded in paraffin. Deparaffinized sections were pretreated with 0.1% trypsin (Sigma-Aldrich) and then blocked with 2% normal donkey serum (Wako). The sections were incubated with an anti-P protein rabbit polyclonal antibody reported previously [42 (link)] and with Alexa Fluor 488-conjugated anti-rabbit IgG (Invitrogen). Nuclei were stained with Mounting Medium with DAPI (Vector, Burlingame, CA, USA). The brain tissues were photographed by using the EVOS M7000 Imaging System (Thermo Fisher Scientific).

The cells in the cell culture chambers were washed with PBS. The cells were then fixed for 15 min with 4% paraformaldehyde (PFA, FUJIFILM Wako Pure Chemical) in PBS and permeabilized for 30 min with 0.1% (v/v) Triton X-100 in PBS. The cells were then incubated in blocking buffer containing 5% normal goat serum (Vector), 5% normal donkey serum (Wako), 3% BSA (Sigma-Aldrich), and 0.1% Tween-20 (Nacalai Tesque, Inc.) in PBS at 4 °C for 24 h. After blocking, the cells were treated with mouse anti-human albumin IgG (Stock solution 10 µg mL⁻¹, diluted by blocking buffer into 20 ng mL⁻¹, Thermo Fisher Scientific) as the primary antibody in blocking solution for 24 h. The following day, after washing the excess antibodies three times with PBS (containing 0.1% Tween-20), the cells were treated with Alexa Fluor 647-labeled donkey anti-mouse IgG H&L (Stock solution 1 µg mL⁻¹, diluted by 0.1% Tween-20 PBS into 1 ng mL⁻¹, Jackson ImmunoResearch) at 25 °C for 1 h. The nuclei of the cells were stained with 50 µL of a solution of 300 nM 4,6-diamidino-2-phenylindole (DAPI, Dojindo Laboratories) at 25 °C for 30 min and then washed twice with PBS.