Localizing Fluorescent Tags to Myelinated Nerve Fibers in Brain Tissue Samples
The rat brain tissue specimen above was fixed, permeabilized, blocked with 10-percent normal goat serum, and then treated with rabbit anti-GFAP (astroglia and neural stem cells) and mouse anti-myelin BP (myelinated nerve fibers) primary antibodies followed by goat anti-rabbit and anti-mouse secondary antibodies (IgG) conjugated to Alexa Fluor 568 and Alexa Fluor 488, respectively. Nuclei in the tissue section were subsequently targeted 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, with the exception of Alexa Fluor 568, which was pseudocolored blue, and Hoechst 33342, which was pseudocolored red.