In a double immunofluorescence experiment, the adherent monolayer culture of owl monkey kidney cells illustrated above was fixed, permeabilized, blocked with 10-percent normal goat serum, and then treated with a cocktail of mouse anti-vimentin (intermediate filaments) and rabbit anti-GPP130 (Golgi complex) primary antibodies followed by goat anti-mouse and anti-rabbit secondary antibodies (IgG) conjugated to Alexa Fluor 568 (pseudocolored blue) and Alexa Fluor 488, respectively. Cell nuclei were counterstained with the red-absorbing dye TO-PRO-3. 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. View a smaller version of this digital image. |