In a double immunofluorescence experiment, an adherent monolayer culture of MRC-5 cells (illustrated above) was fixed, permeabilized, blocked with 10 percent normal goat serum, and treated with a cocktail of mouse anti-vimentin and rabbit anti-PMP 70 (peroxisomal membrane protein) primary antibodies, followed by goat anti-mouse and anti-rabbit secondary antibodies (IgG) conjugated to Cy3 and Alexa Fluor 647 (pseudocolored blue), respectively. DNA in the cell nuclei was counterstained with SYTOX Green. Images were recorded with a 60x oil immersion objective using a zoom factor of 2.5 and sequential scanning with the 488-nanometer spectral line of an argon-ion laser, the 543-nanometer line from a green helium-neon laser, and the 633-nanometer line of a red helium-neon laser. During the processing stage, individual image channels were pseudocolored with RGB values corresponding to each of the fluorophore emission spectral profiles unless otherwise noted above.