Employing Double Immunofluorescence to Label Rat Brain Tissue for Neurofilaments and Glial Cells
Neurofilaments, which are found specifically in neurons and are especially prominent in the axons of the cells, were targeted in a rat brain horizontal section (shown above) with mouse anti-NF-P antibodies followed by goat anti-mouse secondary antibodies conjugated to Alexa Fluor 488. Glial fibrillary acidic protein was simultaneously targeted in the specimen with mouse anti-GFAP antibodies visualized with goat anti-rabbit secondary antibodies conjugated to Alexa Fluor 568. Nuclear DNA was subsequently labeled 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.