The calcium-binding protein calbindin and glial fibrillary acidic protein, a member of the class III intermediate filament protein family, were immunofluorescently labeled in the thick section of rat cerebral cortex shown in the image above by treating the specimen with a cocktail of rabbit anti-calbindin and mouse anti-GFAP primary antibodies followed by goat anti-rabbit and anti-mouse secondary antibodies conjugated to Alexa Fluor 488 (green fluorescence) and Alexa Fluor 568 (red fluorescence), respectively. DRAQ5 (pseudocolored cyan), a DNA-interactive agent that exhibits preferential intercalation at AT base pairs, was utilized to target cell nuclei. Images were recorded with a 40x oil immersion objective using a zoom factor of 1.8 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.