A simple and rapid method for the production of cholera B-chain coupled to horseradish peroxidase for neuronal tracing.

A simple and rapid method is described for the production of cholera B-chain coupled to horseradish peroxidase suitable for neuronal tracing. Horseradish peroxidase was activated with a mild sodium periodate oxidation followed by incubation with the cholera B-chain, to yield the conjugates, samples of which were fractionated further by size exclusion chromatography. Both fractionated material and unfractionated conjugates were tested in vitro by a binding assay on rat synaptic membranes, and in vivo by their ability to undergo retrograde transport following injection into the adrenal medulla of rats. These assays showed that the unfractionated conjugates were equivalent to the fractionated conjugates in binding to the rat synaptic membranes, and that they both provided good retrograde labelling of neurones in the spinal cord after injection into the adrenal medulla, with extensive labelling of the distal dendrites.