Fluoro-Jade: a novel fluorochrome for the sensitive and reliable histochemical localization of neuronal degeneration

Abstract

Fluoro-Jade is an anionic fluorochrome capable of selectively staining degenerating neurons in brain slices. The histochemical application of Fluoro-Jade results in a simple, sensitive and reliable method for staining degenerating neurons and their processes. The technique will detect neuronal degeneration resulting from exposure to a variety of neurotoxic insults. Fluoro-Jade can be combined with other fluorescent methodologies including immunofluorescence, fluorescent axonal tract tracing, and fluorescent Nissl counterstaining. Compared to conventional methodologies, Fluoro-Jade is a more sensitive and definitive marker of neuronal degeneration than hematoxylin and eosin (H&E) or Nissl type stains, while being comparably sensitive yet considerably simpler and more reliable than suppressed silver techniques. © 1997 Elsevier Science B.V. All rights reserved.

Introduction

Many neuroscientists including toxicologists, pathologists, pharmacologists, anatomists and histochemists share an interest in detecting neuronal degeneration. The demonstration of this phenomenon, however, is often difficult from both a technical and an interpretational point of view. Conventional techniques such as H&E (hematoxylin and eosin), or Nissl type stains (crystal violet, thionin, etc.) are technically simple and can be used to infer degeneration based on changes such as neuronal shrinkage, vacuolation, and hyperchromatism 5, 12, 28. Unfortunately, such changes are not necessarily indicative of neuronal degeneration and may be due to processing artifacts or non-lethal alterations in cellular morphology. Processing artifacts can result in both shrunken and hyperchromatic cells 4, 30. Not only are such techniques prone to false positives, but it is also possible to miss degenerating neurons since all cells stain with these dyes while only relatively subtle morphological differences exist between normal and degenerating neurons. These interpretational difficulties make the analysis of conventionally stained material time consuming. In contrast, suppressed silver techniques 6, 9, 11, 18are much better from this point of view in that normal neurons remain unstained while degenerating neurons stain black. The main drawbacks associated with suppressed silver techniques lie in their labor intensive and capricious nature.

Because of the limitations associated with the aforementioned methods, the advantages of a histochemical tracer, which is both simple and reliable as well as highly sensitive and easy to interpret, became apparent and provided the impetus for this research. Toward this goal, we screened a variety of fluorescent anionic dyes under a variety of conditions to characterize the optimal tracer and associated technique for detecting degenerating neurons. The rationale for the choice of dyes and staining conditions is further considered in Section 4. Preliminary work [27]indicated Fluoro-Jade to be the compound most suitable for the detection of neuronal degeneration. The characterization and validation of the Fluoro-Jade method is demonstrated with the use of a number of neurotoxic insults.