Two rapid methods of counterstaining fluorescent dye tracer containing sections without reducing the fluorescence.

A method is described for counterstaining neural tissue containing cells that are retrogradely labeled by fluorescent dyes or horseradish peroxidase (HRP). Specifically, protocols are detailed for the combined use of the tracers with Methylene blue for a Nissl stain or with silver methods for the detection of acetylcholine esterase. The usefulness of these techniques is evaluated in relation to cortico-cortico and thalamocortico projections. The findings indicate that the methods do not mask the labeling of the most sensitive fluorescent dyes or by HRP. Only the yellow dyes are significantly affected by the Methylene blue counterstain. Further, Fast blue labeling in neurons is not significantly diminished by the Bodian fiber stain. The effect of coverslipping sections containing fluorescent dye labeled cells also was evaluated and found to significantly extend the life of the labeling while not reducing the sensitivity. Thus the two counterstaining techniques provide excellent structural information, do not seriously affect tracer labeling and have few of the disadvantages common to other counterstaining methods.