The rat hypothalamus coronal tissue section presented above was immunofluorescently labeled for two different intermediate filament proteins, vimentin and glial fibrillary acidic protein (GFAP). After the specimen was fixed, permeabilized, and blocked with 10-percent normal goat serum, it was treated with a cocktail of mouse anti-vimentin and rabbit anti-GFAP primary antibodies followed by goat anti-mouse and anti-rabbit secondary antibodies (IgG) conjugated to Alexa Fluor 488 and Alexa Fluor 568, respectively. The far-red fluorescent DNA probe DRAQ5 (pseudocolored cyan) was subsequently employed as a nuclear counterstain. Images were recorded with a 40x oil immersion 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 unless otherwise noted above. View a larger version of this digital image. |