Indirect immunofluorescence assay (IFA) to confirm the expression of NiV glycoproteins

RK13 cells cultured in 6-well plates were infected with LC16m8-G or LC16m8-F at an MOI of 0.01. At 40 h post-infection (hpi), the cells were washed three times with PBS and fixed in PBS containing 2% formaldehyde. The cells were then blocked with 1% normal donkey serum (NDS) in PBS (PBS-1%NDS) overnight at 4°C. After removing the blocking buffer, cells were incubated with polyclonal rabbit serum containing anti-NiV G or anti-NiV F antibodies in PBS-1%NDS for 1 h at room temperature [42]. After three washes with PBS, the cells were incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG (H+L) (Invitrogen, Carlsbad, CA, USA) in PBS-1%NDS for 1 h at 26°C. After washing with PBS, stained cells were observed under a fluorescence microscope (Eclipse Ts2-FL).