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Frequently Asked Questions

General | Black Gold | Fluoro-Jade

General

Q>

Background staining occurs before impregnation of all fine myelinated fibers.

A>

Tissue was probably over-fixed. Try to avoid leaving brain in fixative, or sections in buffer, for longer than one month.
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Black Gold

Q>

What is Black-Gold II?

A>

Black-Gold II is a gold-phosphate complex that can produce a high contrast and resolution stain of myelinated fibers.
   

Q>

What is the difference between the original Black-Gold and Black-Gold II?

A>

Black-Gold was first developed as a reliable variant of a gold-chloride based myelin stain. This improvement resulted in a stain that was stable at atmospheric conditions and resulted in consistent staining. Black-Gold II represents an improvement in the synthesis which confirms 3 advantages over the original Black-Gold. Specifically, it results in a high contrast stain without the need to use relatively expensive gold containing intensifiers. It also is easily soluble in saline, unlike its predecessor which required both heating and sonicating to dissolve. Lastly, the more efficient synthesis of Black-Gold II means a 25% more dye per unit price.
   

Q>

Is the formaldehyde fixation necessary?

A>

Yes.
   

Q>

Will the stain work on paraffin or plastic embedded tissue?

A>

No, only non-embedded tissue.
   

Q>

Will Black-Gold stain normal or pathological myelin?

A>

Both. In the normal brain, myelinated tracks appear dark red, while individual fibers appear black. In brains with lesions, the myelinated fibers first appear swollen with conspicuous varicosities. Over time the myelin becomes more fragmented and eventually disappears.
   

Q>

What are the advantages over other myelin stains like Luxol fast blue, or Sudan Black?

A>

Faster, better contrast, better resolution.
   

Q>

Is stained tissue compatible with solvent exposure?

A>

Before Black-Gold II staining tissue, exposure to solvents is counterindicated. After staining however, tissue may be exposed to solvents and their use for dehydration and clearing is appropriate.
   

Q>

Can counterstains be used with Black-Gold II?

A>

Yes, most conventional Nissl stains may be employed for a cytoplasmic counterstain. Red, green or violet colored cellular label will result from the respective dyes neutral red, malachite green or crestyl violet acetate. Following the standard Black-Gold II staining, the slides are transferred to one of the aforementioned dye solutions, typically at a concentration of .1-.4% in water with the pH adjusted to between 3.5 and 4.0. Following 5 min in the stain solution, the slides are transferred through 30 second changes of water, 70% alcohol, 95% alcohol, 2 changes of absolute alcohol and 2 changes of xylene.

Q>

Myelin impregnation incomplete with Black Gold...

A>

Return slides to 60 degrees (C) Black Gold solution and monitor every 3 min under microscope until fine parrallel fibers of layer 1 cortex are visable.
   

Q>

Background has lavender color...

A>

Section has been overstained and staining time should be reduced.
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Fluoro-Jade

Q>

When the background level is too high relative to specific staining...

A>

Leave in fresh potassium permanganate longer, about twenty min...or dilute the Fluoro-Jade concentration by half (.0005%)...Fluoro-Jade B (.0002%).
   

Q>

What is the biological (endogenous) ligand for Fluoro-Jade?

A>

This is not known, although since FJ is a polyanionic fluorescein derivative, it may bind to polyamines.
   

Q>

Can FJ be used with paraffin processed tissue?

A>

Yes. Use xylene to remove paraffin, then rinse twice with alcohol. Also works with cryostat cut un-fixed tissue.
   

Q>

Can it be combined with immunofluorescence?

A>

Yes. Although, sometimes the pretreatment procedures can attenuate immunofluorescent labeling. If so, the time in potassium permanganate solution should be reduced as necessary. Dye concentration may also need to be reduced.
   

Q>

Can FJ be used on unfixed tissue?

A>

Yes, although morphological detail and contrast may be diminished.
   

Q>

Do the FJ dyes only label neurons that die via a specific mechanism?

A>

Thus far, all evidence indicates that the dyes label degenerating neurons, regardless of the mechanism of cell death. Thus FJ dye will label neurons following exposure to neurotoxicants (excitotoxins, metabolic respiration inhibitors, aromatic mono-amide agonists, etc) physical trauma, ischemia and programmed cell death. Therefore it will detect both apoptotic and necrotic cell death. Its usefulness for detecting neurons lost in chronic human degenerative diseases, remains to be evaluated.
   

Q>

Do the FJ dyes stain any brain structures other than degenerating neurons?

A>

Two structures adjacent to the brain that will stain are the choroid plexes and the meninges. In unperfused animals red blood cells will stain. These structures are easy to distinguish from degenerating neurons by their location and morphology. Although more faintly stained than degenerating neurons, some semi-chronic insults will result in localized astrocytic staining as well. These astrocytes typically double label with GFAP suggesting they are activated rather than degenerating.
   

Q>

What are the FJ dyes?

A>

All are analogues of the fluorochrome, fluorescein. Fluoro-Jade contains additional carboxylic groups, Fluoro-Jade B has both phenolic and carboxylic substitutions, while Fluoro-Jade C is the sulfate ester of Fluoro-Jade B.
   

Q>

How do the staining properties differ for each Fluoro-Jade dye?

A>

In general, fluorescence intensity is reciprocally related to tissue affinity. Thus FJ is the most fluorescent dye but exhibits the lowest affinity for degenerating neurons. Conversely, FJ-C is the least fluorescent but exhibits the highest affinity for degenerating neurons. In practice, this means that FJ-B can result in a stain of higher contrast and resolution than FJ, while FJ-C will result in staining of the highest resolution and contrast.
   

Q>

What are the emission and excitation peaks for the FJ dyes?

A>

Emission: 385nm
Excitation: 425nm

In practice, they can be visualized with a microscope equipped with a standard filter for imaging fluorescein or FITC.
   

Q>

Is it possible to counterstain all cells when staining with the FJ dyes?

A>

Yes, incorporation of .0001% DAPI (4',6 Diamidino-2-Phenylindole; Sigma) into the FJ staining solution will result in a blue fluorescent nuclear stain, when visualized with ultraviolet light.
   

Q>

Is it possible to combine FJ labeling with immuno-fluorescent methods?

A>

Yes. This is typiclly accomplished by first processing the loose tissue sections according to standard immunofluorescent methods. A TRITC-conjugated secondary antibody is suitable for multiple labeling with the FJ dyes. The loose immuno-fluorescent labeled tissue is mounted on gelled slides and air dried. Then the slides are rehydrated in water and transferred to the potassium permanganate pretreatment solution. Time in this solution may be reduced should it attenuate the immuno-fluorescent staining. Note: dead neurons may fail to express certain immunological epitopes upon cell death.
   

Q>

What is the endogenous neuro-degeneration molecule that the FJ dyes bind to?

A>

This is not known. Speculation concerning the identity of the endogenous neuro-degeneration molecule has been discussed in the FJ-C manuscript (Brain Res. Vol. 1035/ issue 1/ P. 24-31, Feb. 2005). Putative candidates include polyamines such as putrescine, spermadine, cadaverine and histamine which have all been implicated in peutrification. Alternatively, changes in membrane bound molecules such as phosphotidy/choline or phosphotidy/serine have been implicated in neuronal degeneration. Lastly, proteolytic cleavage of the microtubule protein, C-Tau has also been implicated in neuronal degeneration.
   

Q>

What is the preferred sectioning method?

A>

The FJ dyes are compatible with virtually all sectioning methods including cryostat, vitratome, freezing-sliding microtome, and paraffin microtome. We routinely cut 25um thick sections on the freezing sliding microtome. Paraffin embedded sections are used when tissue sections with a thickness of 10um or less are needed. Thinner sections tend to photograph better, specifically at higher magnifications.
   

Q>

How should the tissue be fixed?

A>

Although the dye can be used on unfixed tissue, the morphology and contrast are better in fixed tissue. Neutral buffered formalin or paraformaldehyde is the preferred fixative, as fixatives containing gluteraldehyde tend to produce autofluorescence, while fixatives containing heavy metals, or the use of iodine to remove them, can quench the dye's fluorescence.
   

Q>

How should the dye be stored?

A>

The dry powder can be stored at room temperature, preferably in a dessicator. The .01% stock solution in distilled water should be stored in a refrigerator. The .0004-0001% working solution in .1% acetic acid should not be stored.
   

Q>

How long can the dye be stored?

A>

The dry powder can be stored indefinately. The stock solution can be stored for at least 2 months in the refrigerator, but should be discarded if the solution becomes turbid. The working solution should be used within 2 hours of preparation.
   

Q>

Can the FJ dyes be used in tissue culture preparations as well?

A>

Yes, essentially the same paradygm for staining tissue slices can be used with only a few modifications. Typically the cells are fixed in situ with 1-2% formaldehyde (2.5-5% formalin) in .1M neutral phosphate buffer, or tissue culture media. After 1 hour fixation, the cells are rinsed with distilled water and then stained according to the standard FJ-C staining protocol, starting with immersion in the potassium permanganate solution. Reagents can be decanted directly into the tissue culture dishes. After staining the dishes are air dried and a coverslip is mounted over the bottom with DPX (Aldrich) mounting media.
   

Q>

Are the FJ dyes susceptable to fading?

A>

In terms of archival storage, the dyes are extremely stable. No loss of fluorescence could be detected in tissue prepared up to 5 years earlier. The dyes are also very stable under epifluorescent illumination. Although no loss of signal could be detected following one hour of constant epifluorescent illumination using 4x or 10x objectives, some loss of signal could be detected following an hour epifluorescent illumination under higher power objectives. This suggests that low magnification photomicrographs should be taken prior to prolonged illumination at high magnifications.
   

Q>

What mounting media should be used?

A>

Mounting media should contain non-polar solvents and be non-fluorescent. DPX (Aldrich) is recommended, although permount can be used. Mounting media containing polar solvents including water, glycerin or alcohol are counterindicated.
   

Q>

What is the optimal post-insult survival interval?

A>

To a large extent, this depends on the nature of the insult. For kainic acid, FJ-B positive cells could first be detected at 3 hours post-dosing. The number and staining intensity of labeled cells increased over the first 24 hours, whereas it remained relatively constant for the next 14 days. Neuronal death from substituted amphetamines was maximal at 1-3 days post-dosing, while damage from metabolic inhibitors of respiration such as 3-nitropropionic is maximal at longer survival intervals such as 5-7 days. A 2 day survival interval is often recommended for preliminary studies.
   

Q>

What is a good positive control?

A>

Kianic acid (10mg/kg,/i.p.) exposure results in reproducable and prominent forebrain lesions of the cortex, hippocampus, and thalamus. Lesions will be found in all animals exhibiting limbic type seizures. Should seizure activity continue for over 2 hours and become life threatening, they can be blocked by the administration of barbituates. A 1-2 day post-dosing survival time is recommended.
   

Q>

Do the FJ dyes detect dead or dying cells?

A>

All evidence indicates that only cells that are dead and undergoing degeneration label, as opposed to cells that have been damaged, but remain viable.
   

Q>

Will the FJ dyes label any type of dead cell?

A>

No, they are specific for dead neurons.
   

Q>

Which dye is most cost efficient?

A>

Although by weight FJ is the least expensive, while FJ-C is the most expensive, it should be noted that FJ is used at a 10 times greater concentration than FJ-C. Therefore in terms of cost per section, FJ-C is the least expensive.
   

Q>

Can the FJ dyes be used with living cells?

A>

Although the dye itself is of low toxicity, the .1% acetic acid vehicle typically precludes its use with living cells.
   

Q>

Is the staining of paraffin embedded tissue different from frozen tissue?

A>

The only difference is in the initial steps in which the paraffin is removed in two changes of xylen, followed by two changes of alcohol and subsequent rehydration through gradated alcohols. The basic ethanol solution is not needed as the xylene will also remove lipids.
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Fluoro-Jade Trouble-Shooting

Problem>

Tissue wrinkles or falls off slides when processing.

Solution>

Use proper slide gelling procedure. This is described in detail in the methods section/see protocol on the Products page.
   

Problem>

Staining present, but low contrast (high background).

Solution>

a) Reduce dye concentration by 50%
b) Increase time in KMnO4 by 5 min
c) Double rinse time
   

Problem>

Staining present, but faint.

Solution

a) Double the FJ concentration
b) Reduce time in KMnO4 by 5 min
   

Problem>

Stain present after final rinse, but lost after coverslipping.

Solution>

a) Air dry slides rather than solvent dehydration.
b) Avoid mounting media that contain polar solvents including water, glycerin, ethanol, etc.
   

Problem>

No staining.

Solution>

a) May be due to the absence of neurodegeneration - verify by running positive control - kainic acid 10mg/kg, i.p.
b) Avoid solvent dehydration, or mounting media containing polar solvents.
c) Make sure stain working solution is in .1% acetic acid.
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