Immunofluorescence with mouse anti-alpha-tubulin was employed to visualize distribution of the microtubule network in the log phase monolayer culture of opossum kidney cells presented above. The secondary antibody (goat anti-mouse IgG) was conjugated to Alexa Fluor 546 and mixed with Alexa Fluor 488 conjugated to phalloidin to simultaneously image tubulin and the actin cytoskeleton. Nuclei were counterstained with TO-PRO-3 (pseudocolored cyan). 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.
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