The blue reaction product in horseradish peroxidase neurohistochemistry: incubation parameters and visibility

A blue reaction product is formed at sites that contain horseradish peroxidase (HRP) activity when benzidene is used as the chromogen. With neutral red as a counter stain, this method affords excellent visualization of both retrograde and orthograde axonal transport of intracerebrally injected HRP. The visibility of this blue reaction-product is better than the visibility of the brown reaction-product obtained in the commonly used diaminobenzidene procedures. Variations in incubation times and reagent concentrations resulted in significant differences in the extent to which transported HRP could be demonstrated with benzidene. One of these benzidene procedures demonstrated a wider extent of HRP transport than a representative diaminobenzidene procedure. The substantia nigra and the nucleus locus ceruleus did not display artifactual deposition of the blue reaction-product.