Brain Sagittal Section Triple Labeled with Alexa Fluor 488, Alexa Fluor 568, and Hoechst 33342

In a double immunofluorescence experiment, rat brain tissue section illustrated above was fixed, permeabilized, blocked with 10-percent normal goat serum, and then treated with a cocktail of mouse anti-BBB (blood brain barrier) and rabbit anti-GFAP (glial fibrillary acidic protein) primary antibodies followed by goat anti-mouse and anti-rabbit secondary antibodies (IgG) conjugated to Alexa Fluor 568 and Alexa Fluor 488, respectively. DNA in the cell nucleus was counterstained with Hoechst 33342. 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.