Streptavidin alexa fluor 568 igg

To visualize the subcellular localization of the transiently expressing POI, cells were plated on coverslips (thickness no. 1.5 and radius: 18 mm). For fixed cell imaging, cells were fixed with 4% paraformaldehyde and permeabilized with cold methanol for 5 min at −20 °C. Next, cells were washed with Dulbecco’s phosphate-buffered saline (DPBS) and blocked for 1 h with 2% BSA in DPBS at room temperature. To detect APEX2-fusion expression, cells were incubated with mouse anti-V5 antibody (Invitrogen, cat. no. R960-25, 1:5000 dilution) for 1 h at room temperature. After washing four times with TBST each 5 min, cells were simultaneously incubated with secondary Alexa Fluor 488-goat anti-mouse IgG (Invitrogen, cat. no. A-11001, 1:1000 dilution) and streptavidin–Alexa Fluor 568 IgG (Invitrogen, cat. no. S11226, 1:1000 dilution) for 30 min at room temperature. Cells were then washed four times with TBST each for 5 min. Immunofluorescence images were obtained and analyzed SP8 X, Leica (NICEM in Seoul National University, Korea) with objective lens (HC PL APO ×100/1.40 OIL), White Light Laser (WLL, 470–670 nm, 1 nm tunable laser), HyD detector, and controlled by LAS X software.