Visualizing His-Tagged Proteins in Sf21 Cells

IFA was performed according to previous studies (20 (link), 24 (link), 27 (link)). Sf21 cells (1 × 10⁴ cells/well) were seeded into the wells of Millicell^® EZ chamber slides (Millipore, PEZGS0816) and infected with individual recombinant baculoviruses at multiplicity of infection (MOI)=1 for 2 days. The infected cells were washed once with DPBS and then fixed with 4% paraformaldehyde (Electron Microscopy Science, 15710) for 15 min at room temperature. After blocking with ChonBlock Blocking/Sample Dilution Buffer (Chondrex, 9068), the cells were incubated with primary mouse anti-His antibody (1:200; Bio-Rad, MCA1396) for 2 h at room temperature. After three washes with DPBS plus 0.1% Tween 20 (DPBST), the cells were incubated with Alexa Fluor 555 Goat anti-Mouse IgG (H+L) secondary antibody (1:5000; Invitrogen, A21422) and counterstained with 49,69-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific, D1306). Distribution of fluorescence signal was observed under Zeiss laser confocal microscopy (LSM780).