Further studies on the use of the fluorescent tracers fast blue and diamidino yellow: effective uptake area and cellular storage sites.

Some basic methodological issues concerning the use of the fluorescent tracers Fast blue (FB) and Diamidino yellow (DY) were studied using the projections of the red nucleus to the nucleus interpositus anterior (NIA) of the cerebellum of the cat. On standard Nissl-stained sections, it was possible to delineate 4 distinct zones at the FB and DY injection sites. Correlative studies of injection sites in the NIA and retrograde labeling of cell bodies in the contralateral red nucleus showed that effective uptake occurred only from the zone mechanically damaged by the injection needle (termed zone 0). The tracer remains in this zone during the post-injection survival. The limited uptake area for both tracers is a valuable feature for studies of restricted neuronal projections. However, the tracers are not suitable for use in quantitative studies, especially those concerning axonal collateralization. Perfusion with water-soluble fixatives did not alter the cellular storage site. In double-labeling experiments using horseradish peroxidase and DY, the HRP histochemistry induced an important "washing out" of DY and consequently, an underestimation of the number of labeled neurons.