Alexa fluor 546 conjugated goat anti rabbit igg h l secondary antibody

Cells were fixed with 2% (w/v) paraformaldehyde for 10 min at 4 °C, permeabilized with 0.1% Triton X-100 in PBS for 5 min, blocked with 3% BSA-PBS for 30 min at room temperature and probed with a cytokeratin pan type I/II antibody cocktail (clone AE1/AE3, Thermo Fisher Scientific, Waltham, MA, USA) at a dilution of 1:1000. FeMV-GT2 was probed using a polyclonal rabbit antibody raised against the nucleoprotein of FeMV (generated in house) at a dilution of 1:100. After overnight incubation at 4 °C cells were washed three times with PBS and incubated with an Alexa Fluor 488-conjugated goat-anti-mouse IgG (H+L) or with an Alexa Fluor 546-conjugated goat-anti-rabbit IgG (H+L) secondary antibody (Thermo Fisher Scientific, Waltham, MA, USA) in 3% BSA-PBS at a dilution of 1:1000 containing 4′,6-diamidino-2-phenylindole (DAPI, 300 nM) and incubated for one hour at 37 °C in a humidified chamber. After three PBS-washing steps images were acquired using an AX70 microscope (Olympus, Tokio, Japan). Virus titration was performed by the endpoint dilution assay in LLC-MK2 cells followed by visualization of virus positive dilutions using immunofluorescence staining. Viral titers were expressed as 50% tissue culture infective dose (TCID₅₀) calculated after the Reed–Muench method [21 ].