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Direct Dyes for Staining Amyloid

The most common methods for demonstrating amyloid depend on it being stained with the dye Congo red (CI 22120). This is an acid dye which stains cytoplasm pink. It belongs to a class of anionic dyes named direct dyes or, more often, direct cotton dyes, because they can stain cotton “directly” in textile use, that is, without using a mordant.

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Applicable Dyes

Congo red was the first direct cotton dye used for demonstrating amyloid and has become something of a reference for the purpose. Other direct cotton dyes have been proposed as replacements but congo red is still the most popular. The most common alternative is sirius red F3B (CI 35780), which is a somewhat darker red than congo red, and is sometimes preferred as being easier to see. There are also benzo scarlet 4BNS (CI 29200), which is sold under the name “Amyloid red” by one laboratory supply company and is similar in color to sirius red F3B, and sirius scarlet GG (CI 40270), which has a slightly yellower tone. There are others as well but they have gained little popularity. In addition to sirius red F3B and sirius scarlet GG, Putchler, Sweat and Kuhns reported on several other dyes, including those capable of staining amyloid red, blue or yellow.

Pseudo-Crystal Structure

Beta sheet dye

The selectivity of direct cotton dyes for amyloid is thought to be due to its β-pleated sheet structure in conjunction with dye attachment by hydrogen bonding along the pleats of the folded protein.

Due to this, the dye is aligned in a very regular pattern. Since one of the characteristics of crystals is that the molecules form regular patterns, this regular attachment of dye is sometimes termed a “pseudocrystal”, i.e. a false crystal. It displays some of the characteristics of a crystal but, of course, is not a real crystal since it is formed from dye molecules attached to protein. Nevertheless, the optical properties of congo red stained amyloid are either caused by the structure of this pseudocrystal or accentuated by it.

Hydrogen Bonding

Putchler, Sweat and Levine showed that the binding of congo red to amyloid was by hydrogen bonds, and since then there have been several modifications to the methods using direct cotton dyes to facilitate staining by this means. Three such modifications are common: the use of a less polar solvent such as ethanol, the addition of alkali to raise the pH, and the addition of sodium chloride. The first two of these tend to reduce ionic bonding of acid dyes to basic tissue components, thus reducing background staining. The addition of sodium chloride is to facilitate the close approach of an acid dye to acid tissue components.

Considerations for Staining

Polarity of the Solvent

As they are a subgroup of acid dyes, it would be expected that direct cotton dyes would bond ionically to basic tissue components, and this is the case. Congo red is rarely used this way in practice, but can stain tissue pink quite well. Sirius red F3B is quite distinctly red and has been recommended in a Van Gieson type procedure for collagen which it stains ionically. The solvent is usually water for these applications, but it is well known that using a less polar solvent reduces ionization and tends to lessen the intensity of staining. So it is that background staining can be paler with direct cotton dyes if ethanol is used as the solvent, permitting amyloid to be seen more easily. Other low polarity solvents may also be useful.

Adjusting the pH

A common form of accentuation is pH control. Usually, acid is added to acid dyes to increase the amount which will attach to basic tissue components, and alkalis added to basic dyes to increase attachment to anionic tissue components. The reverse may also be done, that is, an alkali may be added to acid dye solutions to inhibit ionization and decrease ionic staining. Since hydrogen bonding is not affected by altering pH, this becomes an effective means of suppressing attachment of the dyes to basic tissue components. Usually a pH in the neighborhood of 10 is sufficient, but pH levels lower than this will still reduce the amount of staining considerably.

Presence of Sodium Chloride

A less common means of accentuation of dye staining is the use of so-called “indifferent salts”, a name given to them before their mechanism was understood. When they dissolve these salts, usually sodium chloride, ionize and the resulting solution will contain both positively charged cations and negatively charged anions. Since like repels like and opposites attract, it is to be expected that the positively charged sodium ion will be attracted to negatively charged groups in the tissue, effectively annulling their charge so they can no longer repel negatively charged dye molecules. Due to the elimination of these repulsive forces the dye can closely approach tissue components with either a like charge or an opposite charge, resulting in increased likelihood of hydrogen bond formation should it be possible.

This selectivity for amyloid is not just due to hydrogen bonds between any sites capable of forming them, but involves some steric configuration. That is, the sites on the dye which are capable of forming these bonds must be in appropriate positions relative to the sites in the amyloid. In order for the dye to be deposited in the regular, linear fashion within the folds of the ²-pleated sheet, more than one site capable of forming hydrogen bonds must be involved, or the alignment would be random instead of regular. Puchtler, Sweat and Levine concluded that these binding sites must be 10.3 Ä apart, and that a minimum of two is required. In this way each molecule of dye is attached in the same alignment within the folds of the amyloid sheet forming the pseudocrystal.

Green Birefringence

One of the characteristics of congo red is that the stained amyloid displays “green birefringence”. This is also known as “dichroic birefringence”, dichroic meaning “two colored”, and is often used as an identifying characteristic of amyloid. As it happens, amyloid stained with congo red also displays “dichroism”, and a distinction must be drawn between these two terms since they describe two different phenomena. In practical diagnostic pathology dichroism is not employed much, but the observation of dichroic, or green, birefringence is considered necessary for confirming the identification of amyloid with these dyes. It is unfortunate that the terms are so similar, and perhaps it is advisable to refer to green birefringence rather than dichroic birefringence so as to avoid confusion.

Dichroism requires a single polarizer below the section. With congo red the two colors displayed are colorless and red. Which of these is seen depends on the plane of the light relative to the amyloid, i.e. the phenomenon requires plane polarized light, the colour changing as the plane of the light changes.

Dichroic, or green, birefringence requires two polarizers, one below the section and one above, and the colors displayed are red with the polarizers uncrossed and green when crossed, i.e. the polarizing filter above the specimen detects changes to the plane of polarization caused by the rays passing through the stained amyloid.

So what causes the green color? It is due to an optical phenomenon named “interference”. White light is composed of light waves of many wavelengths which we would see as colored light individually. Mixed together we see white light. Remove any part of the rays and the light appears coloured to us – remove blue get red light for instance. When the congo red stained amyloid causes the plane of light to be rotated it also causes it to be retarded relative to the rest of the light rays, i.e. it becomes out of phase with the rest, much as some light rays do using a phase contrast microscope. In the case of congo red stained amyloid, only some wavelengths are retarded, however. These retarded rays then interfere with the same wavelengths that are not retarded. The effect is to remove some wavelengths, or colours, of light from the white light, causing what is left over and transmitted to be seen as colored light. The color seen is that complementary to what is removed, in this case it appears green.

Sometimes a yellow birefringence is seen as well. This is due to retardation of light rays but is not related to amyloid. It should be ignored.

Staining Methods

Of the methods using direct cotton dyes, Bennhold’s may be the most common, but it is not the best choice. It has the disadvantage of variable differentiation. The procedure involves staining from an aqueous solution, dipping the stained section into lithium carbonate and then into 80% ethanol without rinsing. The lithium carbonate carryover mixes with the ethanol to make an alkaline differentiating alcohol, the strength of which varies depending on the amount of lithium carbonate carried over. It is very easy to overdifferentiate this method, rendering the amyloid excessively pale or uncoloured. Congo red is not an intensely coloured dye to begin with, so a pale result may be difficult to see.

More easily controlled is Highman’s method. It also has the advantage that dyes other than congo red may be used, certainly sirius red F3B and sirius scarlet GG can and it is quite likely that benzo scarlet 4BNS may as well, although the author has had no experience with that last dye. Highman’s method is reliable and is recommended for routine use.

The method generally considered to be the most reliable is Puchtler, Sweat and Levine’s alkaline congo red. The reason it is not more popular is that the working solutions have a short life and must be made up within a few minutes of being used. The other congo red methods are satisfactory and it is a matter of personal choice which is most appropriate.

Both Sweat and Putchler’s sirius red and Llewellyn’s modification of it both work well, and either gives reliable results.

Once again, it should be noted that amyloid is a variably staining material, so more than one technique should be available. It is also recommended that more than one dye be available as well. Congo red and sirius red would be appropriate, using at least one technique with each dye, and a second technique made available for those occasions when a material presumed to be amyloid fails to stain as expected.

These methods stipulate counterstaining with a hemalum solution, usually a progressive type such as Mayer’s. Usually this is after the staining has been done with the direct cotton dye. It is recommended that the nuclei be stained before the amyloid. The alkaline solutions will then adequately blue the hemalum. There may be a slight reduction in amyloid intensity when the nuclei are counterstained after the amyloid is stained, possibly due to the mildly alkaline water wash necessary for blueing.

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  1. Putchler, H., Sweat, F. & Levine., M., (1962),
    On the binding of congo red by amyloid.
    Journal of Histochemistry and Cytochemistry, v. 10, page 355.
  2. Putchler, H., Sweat, F. & Kuhns, J. G., (1964),
    On the binding of direct cotton dyes by amyloid.
    Journal of Histochemistry and Cytochemistry, v. 12, page 900.
  3. Sweat, F. and Putchler, H., (1965),
    Demonstration of amyloid with direct cotton dyes.
    Archives of pathology, v. 80, page 613.