Retrograde neuronal tracing with cholera toxin B subunit: comparison of three different visualization methods

In this report a comparison is made of three different visualization methods of rat cervical motoneurons retrogradely labelled with cholera toxin B subunit (CTb). CTb conjugates such as CTb-HRP and CTb-FITC or CTb-TRITC, which can be visualized after histochemical detection and by fluorescence microscopy, respectively. The following results were obtained. (1) Immunochemical detection of CTb with peroxidase and DAB-Ni incubation provides the best labelling of the cell bodies and their processes, whereas immunochemical detection with FITC produces less effective labelling of the dendrites. (2) Histochemical visualization of CTb-HRP conjugate gives results similar to those of CTb immunochemistry but produces a much more granular appearance of the label, which may affect the identification of distal dendrites. In addition, direct electron-microscopic analysis of labelled structures can be achieved. (3) CTb-FITC and CTb-TRITC visualization permit double-labelling experiments but the labelled cells exhibit fluorescence only in their somata and proximal dendrites. (4) Factors other than labelling intensity, e.g. double-labelling, preservation of the label, compatibility with other techniques and even economic reasons must be taken into consideration when a selection of visualization methods is to be made.