A1rsi laser confocal microscope

We performed immunohistochemistry following previous report^{18 (link)}. In brief, after fixation in 4% paraformaldehyde in phosphate buffer (PB, pH 7.4), the brains (Control: n = 3, HCNP-pp KO: n = 3) were equilibrated in 30% sucrose solution in PB and sectioned at 20 μm by using a cryostat (Leica Microsystems, Bensheim, Germany). The sections were incubated overnight with the anti-HCNP-pp antibody (1:500), the anti-ChAT antibody (1:200), or the anti-VAchT antibody (1:4000) in 1% BSA/PBST at 4 °C. Bound antibodies were detected with an Alexa Fluor 488-conjugated or an Alexa Fluor 594-conjugated donkey anti-rabbit IgG secondary antibody (Thermo Fisher Scientific, Japan). Fluorescent signals were detected with an A1Rsi laser confocal microscope (Nikon, Tokyo, Japan).

A549 cells were seeded on 12-mm coverslips in 24 wells, and siRab5a and siCON were transfected into cells for 36 h. For the binding assay, the transfected (or untransfected control) cells were washed with PBS three times, and then virus (MOI = 10) was added and incubation was performed at 4°C for 10, 30, and 60 min. For the endocytosis assay, the cells were chilled on ice, virus (MOI = 10) was added on ice for 1 h, and then the cells were washed with PBS three times. Complete medium was added and cells transferred to cell incubator for 1 h, and then the plates were transferred onto ice to stop viral entry. Cells were washed three times with PBS to collect the unbound virus and fixed with 4% paraformaldehyde. The cells were then permeabilized with 0.1% Triton X-100 and stained for RSV and F-actin and with DAPI. Images were acquired with a Nikon A1Rsi laser confocal microscope, followed by quantification using the ImageJ software.