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Yellowsolve Staining

A variation on the theme of trichrome staining are what Lendrum and his co-workers referred to as Yellowsolve methods. These are methods in which a small to medium molecular weight red dye is used to stain tissue, then the section is dehydrated and a large molecular weight yellow dye dissolved in a non-aqueous solvent is applied and replaces the red dye progressively. The solvent they used was Cellosolve (2-ethoxy-ethanol). The name applied to the methods is derived from yellow cellosolve.

Protocol Development

These methods are derived from Masson’s HES (Hematoxylin, Erythrosin, Saffron) stain, a technique which used aqueous solutions of erythrosine B and saffron to obtain pink cytoplasm contrasted with yellow collagen. This gave rise to the HPS (Hematoxylin, Phloxine, Saffron) method, in which the saffron was applied in anhydrous ethanolic solution. Still, the results were pink cytoplasm in contrast to yellow collagen. Both of these methods were general oversight stains, designed to compete with an H&E.

Lendrum then used this same principle of a dye in an anhydrous solvent to differentiate and stain collagen with the phloxine tartrazine method for acidophil virus inclusion bodies and paneth cell granules. More involved are methods such as the Slidders’ Fuchsin-Miller for fibrin, and the yellowsolve I and yellowsolve II methods by Lendrum et al. for fibrin and cytoplasmic inclusions respectively.

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The more complex methods may include degreasing, treatment with strong fat solvents such as trichlorethylene and tetrachlorethylene for periods up to 48 hours. In the yellowsolve methods these are an integral component of the techniques. The action of the solvents is inadequately explained, but is usually considered to be due to removal of all protein bound or associated lipids, thus permitting more intimate interaction between the dyes and the proteins.

Some of these methods may incorporate a polyacid (usually phosphotungstic) to differentiate the red dye before applying the anhydrous yellow dye, improving contrast and perhaps depth of staining. The inclusion of the polyacid clearly shows the relationship between these methods and the more common aqueous based trichrome methods, as the fundamental principle is essentially the same, that one acid dye can progressively displace another and does so with the more easily accessed materials, such as collagen, first.

The purpose of the dye solvent is to slow down the process of displacement. The solvents used are of lower polarity than water so, while ionic activity still takes place, it does so at a slower pace. This allows plenty of time for removal of the reagents thus stopping the displacement when the desired degree of color has been achieved.

Although the most common solvent used is 2-ethoxy-ethanol, there is no reason why others could not be employed. In fact, the initial method (HPS) used absolute ethanol. However, it is important that the solvent be anhydrous. All traces of water must be removed. It is important, therefore not only to ensure that the solvent itself does not contain any water, but also to ensure that the section is thoroughly dehydrated before placing it into the dye solution and that moisture does not accumulate during the staining itself. For these reasons, the yellow dye is usually applied in a Coplin jar or other container rather than on a staining rack over a wet sink. In some methods it is even necessary to seal the container in which the solution is applied to inhibit any chance of absorption of moisture from high humidity. The reason for this concern about an anhydrous solvent is that traces of water alter its polarity, and can cause improper displacement as a consequence.

Finally, it should perhaps be noted that, since the last dye is applied in an anhydrous solvent, neither a final wash with water nor dehydration is required. A simple rinse with the solvent to remove excess dye, followed by clearing is all that is required. Indeed, introduction of a water rinse may well remove most of the yellow staining almost instantaneously.