Vinculin, a protein associated with focal adhesion and adherens junctions, was immunofluorescently targeted in a culture of embryonic rat thoracic aorta cells with anti-vinculin mouse monoclonal primary antibodies followed by goat anti-mouse Fab heavy and light chain fragments conjugated to Alexa Fluor 647 (pseudocolored red). In addition, the specimen was stained for mitochondria with MitoTracker Red CMXRos (pseudocolored yellow) and for F-actin with Alexa Fluor 488 conjugated to phalloidin. Cell nuclei were counterstained with Hoechst 33342, which emits blue fluorescence when bound to dsDNA. Images were recorded with a 60x oil immersion objective using a zoom factor of 2.0 and sequential scanning with the 405-nanometer spectral line of a blue diode laser, 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.