Goat anti mouse igg heavy plus

MDCK cells were seeded on a 96-well cell culture plate at a density of 50,000 cells per well. Each respective virus was diluted in 1x minimum essential medium (MEM; Gibco). Cells were infected with a multiplicity of infection (MOI) of 1 overnight. The next day, cells were fixed with 3.7% paraformaldehyde (PFA) for an hour at room temperature. Next, cells were blocked by addition of 100 μL per well of 3% non-fat milk prepared in PBS. The blocking solution was removed and 30 μg/mL of each antibody prepared in 1% non-fat milk in PBS (100 μL per well) was added onto the cells for one hour at RT. Primary antibody was removed and the cells were washed three times with PBS. Hundred micro litre of secondary antibody, goat anti-mouse IgG heavy plus light chain (H + L)–Alexa Fluor 488 (Abcam), which was also prepared in 1% non-fat milk in PBS, was added to the cells afterwards at a dilution of 1:1000 for an hour. Cells were again washed with 100 μL per well of PBS three times. Finally, 100 μL per well PBS was added to prevent drying out of cells. Immunofluorescence was observed using a fluorescent microscope (Olympus IX-70) and images were taken and labelled.

MDCK cells were seeded on a 96-well cell culture plate at a density of 50,000 cells per well. Each respective virus was diluted in 1x minimum essential medium (MEM; Gibco). Cells were infected with a multiplicity of infection (MOI) of 1 overnight. The next day, cells were fixed with 3.7% paraformaldehyde (PFA) for an hour at room temperature. Next, cells were blocked by addition of 100 μL per well of 3% non-fat milk prepared in PBS. The blocking solution was removed and 30 μg/mL of each antibody prepared in 1% non-fat milk in PBS (100 μL per well) was added onto the cells for one hour at RT. Primary antibody was removed and the cells were washed three times with PBS. Hundred micro litre of secondary antibody, goat anti-mouse IgG heavy plus light chain (H + L)–Alexa Fluor 488 (Abcam), which was also prepared in 1% non-fat milk in PBS, was added to the cells afterwards at a dilution of 1:1000 for an hour. Cells were again washed with 100 μL per well of PBS three times. Finally, 100 μL per well PBS was added to prevent drying out of cells. Immunofluorescence was observed using a fluorescent microscope (Olympus IX-70) and images were taken and labelled.