Brain Tissue Sample Triple Labeled with Alexa Fluor 488, Alexa Fluor 568, and Hoechst 33342

Immunofluorescence was utilized to label neurofilaments and astrocytes in a thin section of rat brain tissue. First, the specimen was fixed, permeabilized, blocked with 10-percent normal goat serum, and treated with a cocktail of mouse anti-NF-P (phosphorylated neurofilaments) and rabbit anti-GFAP (glial fibrillary acidic protein) primary antibodies. Then, to visualize the primary targets, the tissue section was treated with goat anti-mouse and anti-rabbit secondary antibodies (IgG) conjugated to Alexa Fluor 488 and Alexa Fluor 568, respectively. Finally, Hoechst 33342 was employed to counterstain 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.