Strata of the Rat Brain Visualized with Double Immunofluorescence and Synthetic Fluorophores
In order to visualize myelin sheaths and astroglia in a rat brain sagittal tissue section (illustrated above), the specimen was immunofluorescently labeled with mouse anti-myelin BP and rabbit anti-GFAP primary antibodies followed by goat anti-mouse and anti-rabbit secondary antibodies conjugated to Alexa Fluor 488 and Alexa Fluor 568, respectively. 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.