Fluoro-Jade
Abstract | Protocol
ABSTRACT:
Fluoro-Jade is an anionic fluorochrome capable of selectively staining degenerative 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.
Compound: Fluoro-Jade
Appearance: Dark red powder with green irridescence
Composition: Aniomic fluorescein salt
Molecular Weight: 445
Solubility: Highly soluble in water, alcohol, weak acids and bases
Purity: No amount of fluorescein or Fluoro-Jade polymer was detected by silica TLC and a solvent system of ethyl acetate/n-propinol/ammonium hydroxide/water (35:35:15:15).
Excitation Peak: 480 nm
Emission peak: 525 nm
Filter system for visualizing: fluorescein/FITC
Storage: The powder should be stored well sealed at room temperature, preferably in a desiccator, due to its hygroscopic nature. The 0.01% stock solution in distilled water should be stored refrigerated (around 5 degrees C). The 0.001% working solution in 0.1% acetic acid should be prepared fresh and not be stored or reused.
Toxicity: Although compound appears to be of low toxicity, it has not been extensively evaluated and therefore routine laboratory caution should be exercised. Not for human consumption.
Original Fluoro-Jade Methods Reference: Schmued, et al, Brain Res. 751 (1997) 37-46.
EXCITATION AND EMISSION PROFILE FOR FLUORO-JADE:
View the graph.
REFERENCE:
L. Schmued, C. Albertson & W. Slikker: FLUORO-JADE; A NOVEL FLUOROCHROME FOR THE SENSITIVE AND RELIABLE HISTOCHEMICAL LOCALIZATION OF NEURONAL DEGENERATION.
FLUORO-JADE HISTOLOGICAL STAINING PROTOCOL:
Brain sections were mounted with distilled water onto gelatin coated slides and dried on a slide warmer at 50 degrees C. The tissue was fully dry within 20 minutes at which time it was immersed in 100% ethyl alcohol for 3 minutes followed by a 1 minute change in 70% alcohol and a 1 minute change in distilled water. The slides were then transferred to a solution of 0.06% potassium permanganate for 15 minutes and were gently shaken on a rotating platform. This solution when kept in a sealed glass container remains usable for a period of about 1 week. The slides were rinsed for 1 minute in distilled water and were then transferred to the Fluoro-Jade staining solution where they are gently agitated for 30 minutes. A 0.01% stock solution of the dye was prepared by dissolving 10 mg Fluoro-Jade in 100 ml of distilled water. The 0.001% working solution of Fluoro-Jade was prepared by adding 10 ml of the stock Fluoro-Jade solution to 90 ml of 0.1% acetic acid in distilled water. The stock solution is stable for several months, while the working solution should be prepared immediately before use. After staining, the sections were rinsed with three 1 minute changed of distilled water. Excess water was drained off, and the slides were rapidly air dried on a slide warmer or with a hot air gun. When dry, the slides were immersed in xylene and then coverslipped with D.P.X. (Alrich Chem. Co., Milwaukee, WI) mounting media. Sections were examined with an epifluorescence microscope using a filter system suitable for visualizing fluorescein or FITC. The resulting slides are quite stable and require no special storage conditions or anti-quench agents. The potassium permanganate pre-treatment further enhances the permanence of the preparation resulting in extremely slow fading, even under high magnification epifluorescent illumination.
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