Use of Retrograde Fluorescent Tracers in Combination with Immunohistochemical Methods

The use of retrograde markers in combination with methods for visualizing transmitters or related substances dates back to the mid-1970s (Ljungdahl et al., 1975), and since then many different combinations have been successfully applied for the tracing of transmitter specific pathways in the brain. Following the introduction of the fluorescent retrograde markers by Kuy-pers and his collaborators (Kuypers et al., 1977), several groups have described the use of these popular dyes in combination with immuno-histochemistry, formaldehyde-induced fluorescence histochemistry and acetylcholinesterase (AChE) staining (Hokfelt et al., 1979, 1980, 1983; Ross et al., 1981; Bjorklund and Skagerberg, 1979; van der Kooy and Hattori, 1980; Van der Kooy and Stembusch, Van der Kooy et al., 1981a,b; Van der Kooy and Sawchenko, 1982; Albanese and Bentivoglio, 1982; Skirboll and Hokfelt, 1983; Skirboll et al., 1983; Sawchenko and Swanson, 1981, 1982; Sawchenko et al., 1982; Snyder et al., 1986; Loewy et al., 1986; Thor and Heike, 1987; Charlton and Heike, 1987). The advantage of immunohistochemistry over the latter two techniques lies in its range. In principle, immunostaining permits visualization of any substance against which an antiserum can be raised. Fluorescent immunomarkers, in addition, can be conveniently combined with fluorescent immunohistochemistry through the use of multiple filters attached to the fluorescence microscope.