Glial fibrillary acidic protein (a type III intermediate filament protein), the calcium-binding protein calbindin, and heavy chain neurofilament subunits were immunofluorescently labeled in the coronal section of rat pons tissue presented above by treating the specimen with a cocktail of mouse anti-GFAP, rabbit anti-calbindin, and chicken anti-NF-H primary antibodies. The primary targets were visualized with goat anti-mouse, anti-rabbit, and anti-chicken secondary antibodies conjugated to Alexa Fluor 568, Alexa Fluor 647 (pseudocolored magenta), and Alexa Fluor 488, respectively. Hoechst 33342 was utilized to counterstain cell nuclei. Images were recorded with a 40x oil immersion objective using a zoom factor of 1.2 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.