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

A sagittal section of rat brain (presented above) was immunofluorescently labeled for phosphorylated neurofilaments with mouse anti-NF-P antibodies followed by goat anti-mouse secondary antibodies conjugated to Alexa Fluor 488. In addition, glial fibrillary acidic protein (GFAP), which is strongly and specifically expressed in various astroglia and neural stem cells, was targeted in the specimen with rabbit anti-GFAP monoclonal antibodies visualized with goat anti-rabbit antibodies conjugated to Alexa Fluor 568. Nuclear DNA 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.