Immunofluorescence Staining of UL41 Proteins

HEK293T cells were transfected with pcaggs-UL41-HA, pcaggs-mUL41-HA or empty vector, collected at 36 hpt, fixed with 4% paraformaldehyde overnight at 4 °C, and permeabilized with 0.25% Triton X-100 for 30 min at 4 °C. The cells were rinsed three times with PBST (containing 0.1% Tween-20), blocked with 5% BSA PBS for 2 h at 37 °C, and then incubated with a mouse anti-HA antibody (MBL, Japan, 1:100), goat anti-mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, and Alexa Fluor 488 (Thermo Fisher Scientific, Meridian Road Rockford, USA, 1:1000). All antibodies were diluted in 1% BSA PBS. Finally, cell nuclei were visualized with DAPI (Roche, Mannheim, Germany). Coverslips were sealed with glycerin buffer, and the cells were visualized using a fluorescence microscope (Nikon ECLIPSE 80i, Japan) [46 (link)].