A method for processing fluorescent labelled tissue into methacrylate: a qualitative comparison of four tracers

A technique for preserving fluorescence in retrogradely labelled neurons embedded in resin was developed. Four retrograde tracers were tested, Fast Blue (FB); Diamidino Yellow (DY); tetramethylrhodamine dextran (fluoro-ruby) (TMRD) and fluorescein dextran (fluoro-emerald) (FD). These tracers were applied to the cut end of the sciatic nerves in rats either by: (a) direct application of tracer crystals, or (b) dipping the nerve into an aqueous solution containing the tracer. Each lumbar spinal cord was removed and dehydrated by one of two methods: (a) conventional alcohol dehydration, or (b) dehydration through a graded series of aqueous methacrylate infiltration solutions (inert dehydration). Specimens were embedded in methacrylate and horizontal sections cut. The location of labelled motoneurons was mapped using a fluorescence microscope. Direct application of tracer crystals labelled more motoneurons than dipping. Fast Blue labelled considerably more motoneurons than tetramethylrhodamine. Labelling by all tracers was retained following methacrylate embedding. Fast Blue and Diamidino Yellow required inert dehydration, while tetramethylrhodamine dextran and fluorescein dextran were preserved using conventional dehydration. These results indicate that tissue labelled with commonly used fluorescent tracers can be processed and embedded in methacrylate, thereby permitting quantitative analysis by modern stereological methods. (C) 1999 Elsevier Science B.V. All rights reserved.