Nis elements basic research microscope imaging software

L2 cells were infected at an MOI of 1 PFU/cell for 8 h with MHV^WT, MHV-MERS^WT, MHV-SC2013^WT, or MHV-HKU5^WT. Cells were washed with Dulbecco’s phosphate-buffered saline (DPBS) (Gibco) and then fixed with 4% paraformaldehyde in DPBS (Gibco). After fixation, cells were permeabilized with 0.1% Triton in DPBS (Gibco) and then blocked with bovine serum albumin. Cells were then stained with mouse monoclonal anti-nucleocapsid (1:200) and rabbit anti-Flag polyclonal (Sigma) (1:2,000) antibodies. After the cells were washed, secondary staining with Alexa Fluor 488-labeled goat anti-mouse (1:400) and Alexa Fluor 594-labeled goat anti-rabbit (1:1,000) antibodies was performed. The cells were washed once more, and the nuclei were then stained with 4′,6′-diamidino-2-phenylindole (DAPI). Stained cells were imaged at a magnification of ×400 on an Eclipse TE2000-U fluorescence microscope (Nikon Instruments, Inc.). Images were acquired using NIS-Elements Basic Research microscope imaging software (Nikon Instruments, Inc.). This was performed at least twice for all viruses.