The calcium-binding protein calbindin and heavy chain neurofilament subunits were immunofluorescently labeled in the rat dentate gyrus tissue section (coronal) shown in the image above by treating the specimen with a cocktail of mouse anti-calbindin and chicken anti-NF-H primary antibodies followed by goat anti-mouse and anti-chicken secondary antibodies conjugated to Alexa Fluor 488 and Alexa Fluor 568, respectively. DRAQ5, a DNA-interactive agent that exhibits preferential intercalation at AT base pairs, was utilized to target cell nuclei. Images were recorded with a 20x objective using a zoom factor of 1.4 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, with the exception of DRAQ5, which was pseudocolored blue.