Figure 5.

Dual-color immunostaining of two antigens was achieved by sequential fluorescent polymerization-based amplification (FPBA) reactions. (A) Human endothelial cells were immunostained for nuclear pore complex protein (NPC) using Nile red nanoparticles (NPs; red), followed by a second round of immunostaining for vimentin using yellow/green NPs (green). (B) As a negative control for NPC staining, the NPC primary antibody was omitted from the antibody dilution buffer during the first staining reaction. (C) As a negative control for vimentin staining, the vimentin primary antibody was omitted from the antibody dilution buffer during the second staining reaction. The nucleus was stained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; purple). The scale bar is 10 microns. Images were taken with a 40× oil objective on confocal scanning laser microscope. Mouse anti-NPC antibody was used at 1:1000 dilution, mouse anti-vimentin antibody was used at 1:5000 dilution, and biotinylated anti-mouse antibody was used at 1:400 dilution.