Immunofluorescent Localization of LCDV in Cells

The FG and HINAE cells were seeded on the cover slips according to the method described by Morel et al. [20 (link)]. Briefly, acid etched circular cover slips were kept in 24-well plates, and 10⁴ cells per well were seeded and incubated to allow cells to attach to the cover slips. After 24 h incubation, the media was removed and the wells were carefully washed with PBS. Then cells were inoculated with 4 TCID₅₀/ml LCDV at 22°C for 2 h. After three washes with PBS, cells were fixed with 4% paraformaldehyde (Sangon Biotech, China) at 22°C for 30 min, followed by incubation overnight with anti-27.8R MAbs (1:5000, 3D9: 2G11 = 1:1, v/v) and rabbit anti-LCDV serum (1:500) at 4°C in a moisture chamber. After washing three times with PBS, the cells were incubated for 1 h at 37°C in the dark with fluorescein isothiocyanate (FITC)-conjugated goat-anti-mouse Ig (Sigma) and Cy3-labeled goat-anti-rabbit (Sigma) at a dilution ratio of 1: 256 in PBS in a moisture chamber. 4, 6-diamidino-2-phenylindole (DAPI, Roche) staining (blue) was used to visualize cell nuclei. Slides were rinsed again, and then mounted with 90% glycerin and observed under a fluorescence microscope.