The introduction of immunological techniques to morphology has enabled the identification of antigens, such as enzymes or structural proteins in various tissues. Most of the studies have been based on lesion experiments, but in order to identify the fibre pathways precisely, a combination of immunocytochemistry and retrograde tracer technique is presented. Two approaches have been used. The first ... [Show full abstract] is to employ horseradish peroxidase (HRP) as a tracer and second is to use fluorescent dyes. The disadvantages of these methodologies are described. The chapter introduces a highly sensitive combination method, using biotinized wheat-germ agglutinin (WGA) and HRP as a retrograde tracer marker. The success of this combined technique for electronmicroscopy has enabled to identify the neurons to which the neuromodulator or neurotransmitter system projects and to determine the origin of the terminals that end on the immunoreactive cells. To obtain good results with the combination of light microscope for immunohistochemistry and retrograde tracing, the substrate for avidin-biotin reaction must be selected carefully.