The purpose of this web page is to foster communication regarding the histological tool Fluoro-Jade (FJ).  We here at UC Davis believe that Fluoro-Jade is an excellent marker for identifying degenerating neurons.  Recently, at the Society for Neuroscience and the National Neurotrauma Society meetings, we discovered a large need for assistance in trouble shooting problems and sharing techniques for staining methods using Fluoro-Jade.  We hope to facilitate communication on the use of FJ through this web site.

First and foremost, we would like to acknowledge Dr. Larry Schmued and his laboratory at the National Center for Toxicological Research for discovering and developing the Fluoro-Jade staining technique.  Most of the information presented on this  web page is the result of continued communication between our lab (Neurotrauma Reserach Laboritories) and his lab.  This web page would not be possible without his continued assistance.  Dr. Schmued has published most of these techniques in the journal of Brain Research (his references are below).
 

THE POWER OF FLUORO-JADE

Fluoro-Jade can be used on almost any tissue section.  From our experience FJ will stain any tissue section type or thickness: i.e. frozen sections, vibratomed sections, cryostat sections, or paraffin embedded sections from 3-50 mm.  The staining protocol takes approximately 90 minutes and little prior histological experience is necessary.  When FJ-stained section are viewed under a fluorescence microscope with a FITC filter, degenerating neurons fluoresce brightly in contrast to neighboring neurons.  The staining protocol we use in our lab is almost exactly like that first described in Schmued et al (1997).
 

HISTO-CHEM INC.

Fluoro-Jade can be purchased from a company called Histo-Chem Inc.

Their contact information is:

1-501-397-2128

Histo-Chem Inc.
P.O. Box 183
Jefferson, AR 72079
 

FLUORO-JADE STAINING PROTOCOL

1)  Tissue sections are mounted onto gelatinized slides and allowed to dry at room temp.

2)  Place slides in staining rack (one slide/slot for even staining).

3)  Immerse in 100% EtOH:  3 min

4)  70% EtOH: 1 min

5)  dH20: 1 min

6)  0.06% Potassium Permanganate (KMnO₄):  15 min shaking gently
            dH20        200 ml
            KMnO4     120 mg (expires 3 days; store at 4^oC)

7)  dH20: 1 min

*****  From this step on, perform as much of the protocol as possible away from light.

8)  0.001% Fluoro-Jade staining solution: 30 min gently shaking (in dark)
            FJ Stock Solution  0.01% (expires in 2 months; store at 4^oC)
                    Fluoro-Jade     50 mg
                    dH20              500 ml

            FJ Staining Solution 0.001% (expires in 1 day)
                    FJ Stock Solution    20 ml
                    0.1% acetic acid      180 ml  (180 ml Acetic Acid in 180 ml dH₂0)

9)  dH₂0: 1 min, X 3 (repeat for 3 total rinses)

10) remove slides from rack, lay flat and dry at room temperature
    (From our experience the best method for drying is to place slides on a paper towel and place under a fan for about 20 minutes or until completely dry.  A hot plate 50^oCwill also work, but I prefer the fan.  Allowing the slides to dry overnight in a drawer at room temp also works well.)

11)  xylene: 2 min X 3

12)  coverslip with DPX (Electron Microscopy Sciences, Inc.) and allow to dry
            (For best preservation of sections, apply a clear coat of nail polish around the edges of the coverslip.  We almost always omit this step, but if you want to preserve your sections for over a year this works well.)  I have some FJ stained sections that are over 18 months old and are still viewable.  This is very rare for fluorescent histochemistry.

Hints:  To protect Fluoro-Jade from photo-bleaching, mix the solutions in dim light.  When the slides are staining, wrap the staining tray in aluminum foil.  When necessary, briefly bring the slides out into the light for a few steps and then quickly put them back into a empty drawer or use another method to protect them from the light.)  Fluoro-Jade is very hardy, and resistant to photo-bleaching, but I always take every step to minimize any exposure to the light.
 

DOUBLE LABELING WITH FLUORO-JADE

When you begin to double label with FJ you will quickly realize that FJ is not the ideal compound for double labeling.  However, with a little manipulation most of the confounds can be overcome.

For double labeling, I always perform the FJ staining last.  So first perform any other immunohistochemistry or molecular labeling (Link to TUNEL Staining Protocol).  After your molecular labeling, perform the FJ staining procedure above with the following modifications.
 

1) Omit the KMnO4 pretreatment.  This will cause a large increase in the background staining.  So the other molecular labels need to be very bright.

2) Stain the sections with 0.0001% Fluoro-Jade at 4oC for 1 hr.

3) Rinse sections in dH20 1 min, X 3.

4) Dry, coverslip, and enjoy your new data.
 

LINK TO TUNEL STAINING PROTOCOL
 
 

I'd be happy to answer any questions about this web page.  Also if you find this site to be useful, drop me a comment.  I'd love to hear from you.
 

Return to Neurotrauma Research Laboritories or  Center for Neuroscience
 

REFERENCES FROM SCHMUED'S LAB

 Schmued, L.; Scallet, A.; Slikker., W.,; Binienda, Z..  Distribution of 3-nitropropionic acid (3-NPA) induced degeneration of myelinated axons and terminals: A combined fluoro-jade and AuCl-3 study. Society for Neuroscience Abstracts, v.22, n.1-3, 1996.:1906  Conference: 26th Annual Meeting of the Society for Neuroscience

Schmued, LC; Albertson, C; Slikker, W Jr.  Fluoro-Jade: a novel fluorochrome for the sensitive and reliable histochemical localization of neuronal degeneration.  Brain Research, 1997 Mar 14, 751(1):37-46.

Schmued, LC; Bowyer, JF.  Methamphetamine exposure can produce neuronal degeneration inmouse hippocampal remnants.  Brain Research, 1997 Jun 6, 759(1):135-40.

Freyaldenhoven, TE; Ali, SF; Schmued, LC.  Systemic administration of MPTP induces thalamic neuronal degeneration in mice.  Brain Research, 1997 Jun 6, 759(1):9-17.

Eisch, AJ; Schmued, LC; Marshall, JF.  Characterizing cortical neuron injury with Fluoro-Jade labeling after a neurotoxic regimen of methamphetamine.  Synapse, 1998 Nov, 30(3):329-33.

Schmued, L; Slikker, W; Clausing, P; Bowyer, J.  d-Fenfluramine produces neuronal degeneration in localized regions of the cortex, thalamus, and cerebellum of the rat.  Toxicological Sciences, 1999 Mar, 48(1):100-6.

Schmued, L. C.; Slikker, W.,; Wang, G. J..  Fluoro-Jade B: A bis homolog of Fluoro-Jade with improved degenerate neuron staining properties. Society for Neuroscience Abstracts, v.24, n.1-2, 1998.:1064.  Conference: 28th Annual Meeting of the Society for Neuroscience, Part 1

 Kristensen, B. W.; Noraberg, J.; Jakobsen, B.; Gramsbergen, J. B. P.; Zimmer, J..  Regional vulnerability of organotypic corticostriatal slice cultures to non-NMDA agonists detected by propidium iodide, Fluoro-jade and LDH release. Society for Neuroscience Abstracts, v.24, n.1-2, 1998.:465. Conference: 28th Annual Meeting of the Society for Neuroscience, Part 1

Schmued, L.; Slikker, W.; Bowyer, J..  Demonstration and localization of  D-fenfluramine induced neuronal degeneration in the rat: A Fluoro-Jade Study. Society for Neuroscience Abstracts, v.23, n.1-2, 1997.:275. Conference: 27th Annual Meeting of the Society for Neuroscience, Part 1
 

OTHER FLUORO-JADE REFERENCES

Hornfelt, M.; Edstrom, A.; Ekstrom, P. A. R..  Upregulation of cytosolic phospholipase A2 correlates with apoptosis in mouse superior cervical and dorsal root ganglia neurons. Neuroscience Letters, v.265, n.2, April 16, 1999.:87-90.

Kanthasamy, Anumantha G.; Yun, Richard J.; Nguyen, Bang; Truong, Daniel D..  Effect of riluzole on the neurological and neuropathological changes in an animal model of cardiac arrest-induced movement disorder. Journal of Pharmacology and Experimental Therapeutics, v.288, n.3, March, 1999.:1340-1348.

Noraberg, Jens; Kristensen, Bjarne Winther; Zimmer, Jens.  Markers for neuronal degeneration in organotypic slice cultures. Brain Research Protocols, v.3, n.3, Jan., 1999.:278-290.

Kokaia, Zaal; Andsberg, Gunnar; Yan, Qiao; Lindvall, Olle.  Rapid alterations of BDNF protein levels in the rat brain after focal ischemia: Evidence for increased synthesis and anterograde axonal transport. Experimental Neurology, v.154, n.2, Dec., 1998.:289-301.

Eisch, Amelia J.; Marshall, John F..  Methamphetamine neurotoxicity: Dissociation of striatal dopamine terminal damage from parietal cortical cell body injury. Synapse (New York), v.30, n.4, Dec., 1998.:433-445.

Bowyer, John F.; Peterson, Steven L.; Rountree, Robert L.; Tor-Agbidye, John; Wang, Guang Jian.  Neuronal degeneration in rat forebrain resulting from D-amphetamine-induced convulsions is dependent on seizure severity and age. Brain Research, v.80, n.1, Oct. 26, 1998.:77-90.
 

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