Employing Immunofluorescence to Label Rat Brain Tissue for Myelin CNPase and Glial Fibrillary Acidic Protein

Glial fibrillary acidic protein and cyclic nucleotide phosphodiesterase were immunofluorescently labeled in the rat brain tissue section presented above by treating the specimen with a cocktail of rabbit anti-GFAP and mouse anti-myelin CNPase primary antibodies followed by goat anti-rabbit and anti-mouse secondary antibodies conjugated to Alexa Fluor 568 and Alexa Fluor 488, respectively. Hoechst 33342, a DNA-interactive agent, was utilized to target cell nuclei. Images were recorded in grayscale with a 12-bit digital camera coupled to a Nikon Eclipse 80i microscope equipped with bandpass emission fluorescence filter optical blocks. During the processing stage, individual image channels were pseudocolored with RGB values corresponding to each of the fluorophore emission spectral profiles.