Fitc conjugated anti rabbit antibody

The methodology was previously described (Nishihara et al., 2016; Sano et al., 2010). A polyclonal rabbit anti‐claudin‐5 antibody (1:500 dilution; Zymed) was used as the primary antibody, and a FITC‐conjugated anti‐rabbit antibody (1:3,000 dilution; Thermo Fisher Scientific) was used as the secondary antibody. Fluorescence was detected using a fluorescence microscope (BZ‐9000; Keyence, Osaka, Japan).

Huh7 and SW13 cells were infected with the CCHFV in triplicate, as described by us previously (Appelberg et al., 2020 (link); Krishnan et al., 2021 (link)). Briefly, Huh7 cells were infected with CCHFV IbAr10200 at a multiplicity of infection (MOI) of 1. After 1 hr of incubation (37 °C, 5% CO₂) the inoculum was removed, the cells were washed with PBS, and 2 ml DMEM supplemented with 5% heat-inactivated FBS was added to each well. Samples were collected in triplicate at 24 and 48hpi along with controls. Due to high permissiveness, we restricted the SW13 infection of 1 MOI for 24 hr only. The infection in Huh7 24hpi was confirmed by immunofluorescence staining of CCHFV nucleoprotein-protein. The cells were fixed in ice-cold acetone-methanol (1:1) and stained using a rabbit polyclonal anti-CCHFV nucleocapsid antibody (home-made) followed by a fluorescein isothiocyanate (FITC)-conjugated anti-rabbit antibody (Thermo Fisher Scientific, US) and DAPI (Roche, US).