Technical considerations on the use of horseradish peroxidase as a neuronal marker

Abstract Techniques are described for increasing the reliability and sensitivity of retrograde labeling of neurons with horseradish peroxidase. Labeling is increased by injecting peroxidase where axons are severed. Exclusive use of glutaraldehyde as a fixative and shortening the time used to infiltrate tissue with cryoprotectives are recommended to reduce degradation of injected peroxidase. Localization of the reaction product is facilitated by gallocyanin counterstaining, by use of phase-contrast optics, and by preincubation treatment of tissue in cobalt chloride. The use of diaminobenzidine and its safe disposal are considered.

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