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Histological stains FAQ - H & E Eosin staining


StainsFile

H&E Eosin staining FAQ

 

 


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How many types of eosin are there?

First we should define what is meant by the name eosin. Quite obviously at its simplest it refers to the dyes actually named eosin, particularly eosin Y ws, which is nearly always what is meant, but also to eosin B and ethyl eosin. That is, however, a very restrictive use of the word. There are other dyes which are very close chemical and structural relatives of the three mentioned above. The term eosins (plural) is sometimes used as a group name to refer to all of these dyes, instead of identifying individual compounds. They are all halogenated derivatives of fluorescien and are acid dyes shaded pink to red.

 
Common name C.I. Number
Eosin Y ws 45380  
Ethyl eosin 45386  
Eosin B 45400  
Phloxine B 45410  
Erythrosin B 45430  
Rose Bengal 45440  
Mercurochrome  


 
Eosin Y Ethyl eosin Eosin B
Eosin Y ws Ethyl eosin Eosin B
     
Phloxine B Erythrosin B Rose bengal
Phloxine B Erythrosin B Rose bengal


The last dye on the list, mercurochrome, also contains a significant amount of mercury and is poisonous. It should be avoided. In the past it was used to mark tissues for orientation before paraffin processing. Red tattoo ink from your local tattoo parlor does a far better job and is a lot safer, just don't get it in cuts and scratches.

Reference
Conn's Biological Stains

R. D. Lillie.
Williams & Wilkins, Baltimore, MD., U.S.A.

 


What is the best way to stain with eosin?

The dye usually specified for the H&E is eosin Y. Its popularity is due to its ability to clearly demonstrate different structures by differences in the intensity with which they are stained. In a well stained section these differences appear to be different shades of pink, although they are not.

 
Tissue component Expected results
Erythrocytes
Eosinophil granules
Very bright pink
 
Muscle
Cytoplasm
Deep pink
 
Collagen Pale pink


While the actual colour depth may vary, the comparative differences between the various tissue elements should be present. Other eosins are not as good as eosin Y at producing these differences, although ethyl eosin and eosin B come close. Phloxine B, erythrosin B and rose bengal tend to be more homogenous in their staining as well as being somewhat redder in shade. These dyes can be applied from aqueous or alcoholic solution.

Aqueous
Aqueous solutions are usually used at concentrations of 1%-2%, and are usually made with tap water rather than distilled water. Suitable tap water is hard and mildly alkaline, and there have been strong suggestions that it should contain calcium (and perhaps manganese) ions. Soft, acid tap water is not suitable as the intensity effects are not usually seen, and the staining appears flat and homogenous. These effects are due to the low pH of the water which increases dye attachment to basic (amino) groups causing overall staining to be more intense. A mildly alkaline pH has the opposite effect, i.e. it depresses staining ever so slightly thus maximising differences between the tissue elements. Why calcium is needed has not been explained, other than its salts are the cause of hardness and mild alkalinity.

After applying the eosin solution for a time (1-3 minutes), the sections are rinsed with tap water to remove excess dye. This step should be carefully controlled as the eosin is progressively extracted. Too little diminishes the differential effect, and too much leaves the tissues too pale. The optimal appearance is a matter of personal preference, and should be set in conjunction with the person who will be examining the sections.

Alcoholic
Alcoholic solutions vary from 0.1%-0.5% and the alcohol may vary from 60%-100% ethanol. It is tempting to speculate that the reason for the very wide variations in the concentration of the alcohol are really attempts to control the degree of ionisation of the dye by modifying the polarity of the solvent, i.e. the more water present the more polar the solvent and the deeper the staining.

The type of alcohol is sometimes raised as a query. Industrial grade ethanol (95% or absolute) or industrial methylated spirits are usually satisfactory. The use of reagent grade ethanol is usually not necessary. This is, of course, provided the industrial grade ethanol used is not contaminated with something that might interfere with staining. In more than 35 years, I have encountered this only once in a single 45 gallon drum. If your eosin staining unexpectedly deteriorates, do check this as a source of the problem.

At its simplest, I have used 0.1% eosin Y in 95% or absolute ethanol applied for 15 seconds or so, briefly rinsed with absolute ethanol for about 5 seconds, then cleared. The results were quite satisfactory. The same solution can also be used to stain for longer (1-2 minutes), then washed with ethanol for longer (1-5 minutes) to extract dye in a similar manner as aqueous staining. This works well. I have also used a very complex solution involving the dilution of strong stock solutions in absolute ethanol with weaker ethanol and acetic acid. This works well too.

Acetic acid
A common practice, particularly in North America, is to add acetic acid to the eosin solutions, both aqueous and alcoholic. The amount added can vary considerably from one drop glacial acetic acid per litre to 0.5 mL per litre. The effect of the acid is to increase ionisation of tissue amino groups and results in more dye attaching. Eosin is, after all, an acid dye and responds to the pH of its environment as any other acid dye does. Addition of a small amount of acetic acid can, sometimes, improve eosin staining by increasing the depth of colouration. The differential effect is not always lost when this is done, although it is invariably diminished to a greater or lesser degree.
Like many things, although a little may be beneficial a lot may be a disaster. Too much acid causes intense but flat and homogenous staining and should be avoided, unless that is the effect preferred. It should also be noted that the addition of acetic acid to aqueous solutions of eosin results in essentially the same staining as would be obtained with soft, acid tap water.

Mixing dyes
Although eosin Y is the usual dye used, it is not uncommon for small amounts of these other eosins to be added to the staining solution. A very small amount of phloxine B added to simple alcoholic eosin Y, for instance, causes erythrocytes to become very brightly prominent. You should experiment with the effects, but be conservative with the amounts added otherwise the eosin Y staining will be overpowered.

Recommendation
Rather than concentrate on looking for that special, magical formula which will take care of all the technical difficulties for you, it will be far more effective to learn how to control eosin staining from first principles. Formulas for eosin solutions vary tremendously, but nearly all are used effectively in some place, and nearly all fail in some place. The difference is in the way they are controlled, and that aspect is completely dependent on the histotechnologist.

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Why do my sections look like a red blob after eosin?

One of the effects of adding excessive amounts of acetic acid to solutions of eosin is to reduce or eliminate the differential effect obtained with sections stained with that dye. In extreme cases this appears as an undifferentiated, completely homogenous and flat, deep pink stained mass. It is also possible, in very bad cases, that the nuclei will be red-brown instead of purple-blue. The resolution is to reduce or eliminate acetic acid from the eosin, and use an alternate means of intensifying staining. Increasing the dye concentration or increasing the length of time for which it is applied come to mind. See the previous section.

Fixation can be the cause of this kind of staining. Different fixatives leave tissues more receptive to some dyes than to others. Some depress staining with acid dyes, some greatly increase it. If you have introduced a new fixative, or used one for a special case, you could get this kind of effect. Check the published characteristics of the fixative used to see if this is the cause. If so, you must decide whether the reason for using the fixative outweighs the poor eosin staining, or whether a separate staining protocol for tissues fixed with that solution is necessary.

Poor and inadequate fixation may also cause poor eosin staining. If tissues are not adequately fixed before processing begins they may not be protected from the effects of dehydration and clearing. The ethanol commonly used as a dehydrant is a fixative in its own right. It is also often warmed in modern processors, thereby increasing its fixation effects and artifacts. Unfixed tissues bathed in hot ethanol for several hours may well exhibit all the poor characteristics that process would infer, including excessive pink staining with eosin. The remedy is to fix the tissues before processing them, even if that means using hot formalin. Hot formalin causes less artifact than hot ethanol and is preferable (but, of course, should be avoided if at all possible).

Excessive decalcification is a further cause of this artifact. By excessive is meant that the decalcifying fluid is being applied for too long, the acid is too concentrated, it is being applied at too high a temperature or on unfixed tissue. More details are found in the decalcification FAQ.


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Why are my sections colourless after eosin?

In the same way that the presence of acid can intensify eosin, the presence of alkali will diminish or completely eliminate it. The reasons are much the same, but in reverse. Alkalis cause elevated pH levels, which diminish ionisation of amino groups and inhibit attachment of the dye. The extent to which it does this varies with the degree to which the pH is changed.

The most likely source of alkali is the blueing agent. Many laboratories use either dilute ammonia or saturated lithium carbonate to blue alum hematoxylin stains. When these agents are used, it is imperative that the slides be completely immersed in the wash water for long enough to completely remove all traces of the blueing alkali. If even the smallest trace is left, it will dissolve in the eosin solvent and affect the staining. Special attention should be paid to this point, both as regards total immersion of the slides and the length of time washing is done.

Remember also that the concentration of alkali is cumulative, so while it may not impact too much initially, repeated small transfers will eventually seriously diminish staining. A clue to this is gradual deterioration of staining, rather than a sudden impact.

While alkali from the blueing solution is the commonest source of the problem it is not exclusively so. There have been several reports of contaminated alcohols. Whether these alcohols are used as the solvent for the eosin or only as the dehydrant after staining does not seem to matter too much. They can cause serious deterioration of stain intensity. This is a relatively uncommon problem, but if you are certain that alkali from the blueing step is not the problem it would be worth while checking this out.

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What is eosinol, and what use is it?

Eosin is the sodium salt of eosinol. Similar compounds exist for the other eosins as well. It is quite simple to make, but can be purchased. It may be named eosin Y, spirit soluble. Make sure you do not confuse this with ethyl eosin which is also called by that name. They are two different compounds.

 
Fluorescien   Eosinol Y   Eosin Y
Fluorescien   Eosinol Y   Eosin Y


Making eosinol

  1. Prepare a strong solution of eosin Y, about 10% or so.
  2. Add concentrated hydrochloric acid to precipitate eosinol.
  3. Continue until the solution is almost colourless.
  4. Filter and collect the precipitate.
  5. Wash the precipitate numerous times with distilled water.
  6. All traces of hydrochloric acid must be removed.
  7. Dry the precipitate at room temperature.
  8. Grind to a powder with a pestle and mortar.
  9. Store in a tightly capped container.

Eosinol is soluble in ethanol and xylene. It has been recommended for staining difficult tissues, although I have not found it particularly useful. To use, simply dissolve some of the eosinol into one of the dehydrating absolute ethanols, or into the first of the xylenes used for clearing prior to coverslipping. Determine the amount by trial and error. Leave sections in this until they are stained adequately, then finish off the sequence and coverslip.


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  Created September 1997


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