Requirements for obtaining unbiased estimates of neuronal numbers in frozen sections.

The use of frozen sectioning is a convenient and rapid means of observing the results obtained using fluorescent retrograde tracers. Quantitation of these results using the biased stereological methods currently available can be associated with large errors. A recently developed stereological tool, the optical dissector, provides unbiased and efficient results, however, the requirements for its use in frozen sections has not previously been established. In this study, a comparison was made of neuron numbers, estimated using the optical dissector method, in the motoneuron pool retrogradely labelled from the rat sciatic nerve with either Fast Blue or Tetramethylrhodamine dextran (fluoro-ruby) in methacrylate embedded and frozen spinal cord specimens. Despite over 50% shrinkage in the frozen sections, compared with virtually no shrinkage in the methacrylate sections, no significant difference in labelled motoneuron numbers was observed, provided this shrinkage was taken into account. Correction for section shrinkage is therefore essential in order to use the optical dissector with confidence to count fluorescent labelled neurons in frozen tissue.

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.