Differential retrograde labelling with horseradish peroxidase (HRP) and Lucifer yellow (LY) in an invertebrate nervous system--HRP fluorescence and LY preservation limit choice of fixative.

The possible application of the combination of horseradish peroxidase (HRP) and Lucifer Yellow CH (LY) for differential retrograde labelling of a relatively large invertebrate nervous system requiring the enzymatic marker to be processed after fixation and sectioning was evaluated. In control experiments an HRP fluorescence induced by glutaraldehyde (GA) fixation was detected and its spectral properties were investigated. This fluorescence, however, does not occur after previous treatment of the tissue with depolymerized paraformaldehyde (PFA) instead of GA. PFA as a fixing agent is also necessary to yield maximum preservation of LY labelling during subsequent HRP histochemistry. Both findings strongly recommend the use of PFA instead of GA as fixative for the combination of HRP and LY. It is shown that the HRP/LY differential labelling technique renders a valid method for the study of the structural interactions at the fibre level of identified neurones in a relatively large invertebrate nervous system, also after retrograde tracing.