ACTA HISTOCHEM. CYTOCHEM. Vol. 3, No. 1, 1970

A MODIFIED METHOD OF THE REACTION FOR THE HISTOCHEMICAL DEMONSTRATION OF , AND ITS APPLICATION TO THE COLLOIDAL IRON REACTION OF ACID MUCOPOLYSACCHARIDES.

SHUNTAHIROSE, MASAYUKIYASUTOMI, NOBUHIROMURAI, RYOTAROYAMADA, ATSUSHIKATAYAMA, MOTOYOSHI TSUJINO, ZENJI IWASA and A.K.A. RAZZAQ.

Department of Surgery, Osaka UniversityMedical School, Osaka. Receivedfor PublicationJanuary 21, 1970.

In the Prussian Blue method for the histochemical demonstration of iron, some parts of produced pigment are considerably washed away during the dyeing process, because the pigment is water soluble. Consequently decrease and diffusion of the pigment occur and staining of the surrounding tissues take place. The color reaction was carried out in 70% acetone solution in order to decrease the of the pigment. Obtaining the sharp and clear demarcation, this method was applied successfully to the colloidal iron reaction of acid mucopolysaccharides and very strong and clear reaction was obtained.

In all histochemical methods to demonstrate iron, the Prussian Blue reaction is the best one. This method was described by Pearls in 1866 for the first time in which the iron is detected as Prussian Blue. This reaction combining with and Fe+++ is very sensitive, on the other hand nearly all parts of iron exist as a ferric type in vivo. Therefore, the Prussian Blue reaction is thought to be an excellent method in demonstrating iron of tissue. But because the Prussian Blue produced in the reaction is water-soluble and distilled water is used as the solvent of the reagents in his method, the Prussian Blue is easily washed away into the solvent or into the washing water. Therefore, it is difficult to prove the small amount of iron in tissues, and the Prussian Blue diffuses in tissues and the sharpness of the demarcation decreases.

METHOD

A) Solubility of the Prussian Blue.

Four series of 8 test-tubes were prepared. Two series of them contained, each, 10 ml of solution diluted by distilled water in 0, 30, 50, 60, 70, 80, 90, 100%; the one series was added 0.1 ml of HCI and the other was not. In another two series acetone was contained instead of ethanol. Then in each test-tube 20 mg of the Prussian Blue was added and the tubes were shaked and left for 3 hr at room temperature (20•Ž). The solubility of the Prussian Blue was thus investigated.

B) Solubility of potassium ferrocyanide.

Even if it was found that the Prussian Blue was hardly soluble in ethanol or in acetone solution as the result of the experiment A, it was thought that the reaction would hardly occur, if the potassium ferrocyanide, the chief reagent of the reaction, was hardly soluble in the solvent. Therefore, it was necessary to investigate the

18 MODIFIED METHOD OF PRUSSIAN BLUE REACTION 19 solubility of the potassium ferrocyanide in ethanol and acetone of above mentioned concentrations. It was clarified after the experiment A), its detail will be shown in the section of result, that Prussian Blue was very soluble in ethanol solution, but hardly soluble in acetone of high concentration. Therefore, 10% potassium ferrocyanide solution was dripped into distilled water and above described acetone solutions untill the crystal of the potassium ferrocyanide was formed. The solubility was determined according to the quantity of the potassium ferrocyanide solution used. In the other series of acetone solutions, in which conc. HCI added up to 1% finally and the solubility of potassium ferrocyanide was also tested in the same man- ner.

C) Color reaction on filter paper.

If the solubility of the Prussian Blue will be decreased in the acetone solution, the solubility of the Prussian Blue in specimens also will be expected to be decreased and good demarcation will be expected. In order to study the slight difference of the produced by slight changes of the concentration of solvent, the color reaction was performed on the spots of Fe+++ on filter paper. For this purpose No. 51 filter paper For Chromatographic Analysis of Toyo-Roshi Co. Ltd. was used. Spots were placed on filter papers with 0.001%, 0.005%, 0.01%, 0.025%, and 0.05% ferric chloride water solution. After the spots on filter paper dried, they were immersed in the 1% hydrochloric acid acetone solution of variable concentrations which were saturated with potassium ferrocyanide and left for 20 min at room temperature (20•Ž). The index of the sensibility was determined by the concent- ration of ferric chloride of spots which showed the minimum color in each concentra- tion of acetone solution, and the index of the diffusion, with size and density of the corona and sharpness of the spots.

D) Detection of iron in tissues.

We did this experiment in order to know whether the same results would beseen in the practical specimens. We used livers of dogs injected with 6MP, fixed in 10% formalin, embedded in paraffin and cut at 6ƒÊ. And the slides were immersed in distilled water and in acetone of variable concentration. We did not use alcohol solution because the Prussian Blue is very soluble in alcohol. The reason of using such material is that it is easy for us to study the reaction as the liver tissue is homo-

genous and when injected with 6MP, hemolysis, liver cell disturbance and possession of hemosiderin in liver cell are seen. Counterstain was performed by Feulgen reaction before staining iron.

E) Detection of mucopolysaccharides.

The coloring procedure of the colloidal iron reaction of acid mucopolysaccharides is the same as the Prussian Blue reaction which has been utilized to demonstrate the naturally existent iron in the tissues. If the Prussian Blue reaction will be improved, it can be applied to demonstrate acid mucopolysaccharides. In detecting iron in tissues by the method described above, we obtained good results when the reaction 20 HIROSE, YASUTOMI, MURAI, YAMADA, KATAYAMA, TSUJINO, IWASA AND RAZZAQ

was done in high concentration acetone. Therefore, we used 70% acetone in the

procedure of coloring of iron in detecting acid mucopolysaccharides. Its details will be shown in the section of result. There are two different methods of colloidal iron reaction of acid mucopoly- saccharides according to the procedure of producing the colloidal iron. One is a dialysis method (Rinehart's method) and the other is a boiling method (Mowry's method). Both methods were compared in high concentration acetone and in distilled water. The stomachs which were surgically resected were used as materials because they contain of several kinds of acid mucopolysaccharides. They were fixed in 10% formalin and cut at 6ƒÊ. In order to know what kind of mucopoly- saccharide the coloring material is, and what kind of mucopolysaccharide is demon- strated more sharply in high concentration acetone than in distilled water, we made the digestive examination with diastase, hyaluronidase and sialidase after the deparaffinizing procedure was finished. The counterstain was done by Feulgen reaction before slides were immersed in the colloidal iron solution.

RESULTS A) Solubility of the Prussian Blue. The Prussian Blue used in the experiment was completely dissolved in distilled water as shown in Fig. 1. So was it even in 100% ethanol. The solubility decreased in acetone solution. Though it was fairly well soluble in 30% acetone solution, it was hardly soluble in acetone solution of higher concentration. In 50% acetone solution precipitation of the Prussian Blue increased remarkably, in 70% the Prus- sian Blue was almost insoluble, and in concentration higher than 80% it was completely insoluble. (Fig. 2) The Prussian Blue was dissolved better in 1% hydrochloric acid acetone solution than in acetone without HCI. But even in existence of HCl it was hardly soluble in the acetone solution of concentration higher than 70% .

Fig. 1 Fig. 2 B) Solubility of the potassium ferrocyanide. The potassium ferrocyanide was also hardly soluble in high concentration acetone. In 1% hydrochloric acid acetone solution, the solubility of the potassium ferrocyanide increased remarkably. With HCI even in 70% acetone solution, 1.3% of the potassium ferrocyanide was dissolved (Fig. 3). MODIFIED METHOD OF PRUSSIAN BLUE REACTION 21

Fig. 3

But when the concentration of acetone became higher than 90%, the solubility of the potassium ferrocyanide decreased remarkably and only 0.3% of potassium ferrocyanide was dissolved even with HCI.

C) Color reaction on filter paper. Almost same reactions were observed in distilled water and ethanol. No reaction was demonstrated in them when the concentration of ferric chloride was under 0.025%. Even when the concentration was over 0.025%, the outline was not sharp and blue diffusion was seen. There was no difference between the reactions in distilled water and in 1% hydrochloric acid acetone solution of low concentration saturated with potassium ferrocyanide (Fig. 4). When the concentration of acetone became high, the reaction was clearly seen. In 70%-80% acetone solution, even 0.005% ferric chloride was demonstrated clearly, moreover the outline was clear and the diffusion was hardly observed. When the concentration of acetone was over 90%, the reaction was hardly seen. D) Detection of iron in tissues. Experimental study was performed on dog livers treated with 6MP and ex- cellent results were obtained when 70 %-80 % acetone solution was used. We obtained slightly better results in 60% acetone than in distilled water, but the difference was not so remarkable. In Fig. 5 the specimen was stained in distilled water as usual. In Fig. 6 it was stained in 75% acetone solution. In the latter specimen, the reaction was seen more clearly and the demarcation was sharper than those in distilled water. The Prussian Blue was seen in spot-like stain scattering in the liver lobules. Figures 7 and 8 show the specimens in high magnification, 22 HIROSE, YASUTOMI, MURAI, YAMADA, KATAYAMA, TSUJINO, IWASA AND RAZZAQ

Fig. 4

Fig. 5 Fig. 6

Fig. 7 Fig. 8 MODIFIED METHOD OF PRUSSIAN BLUE REACTION 23

which were stained in distilled water and in acetone . In the specimen stained in acetone solution, clear Prussian Blue reaction is seen not only in the stellated cells of Kupffer, but also in the cytoplasm of the liver cells and in the bile canaliculi But in the specimen stained in distilled water , such fine reactions are not seen. E) Detection of mucopolysaccharides . Using distilled water as the solvent to stain iron bound acid mucopolysac- cahrides, the Rinehart's method and the Mowry's method were compared . In the Rinehart's method, the Prussian Blue was seen mainly in goblet cells of intestinal metaplasia and its contrast was clear (Fig. 9). But in the Mowry's method, the fairly strong color was seen in connective tissues besides metaplasia and the color apparently seems to be due to the deposit of iron (Fig. 10).

Fig. 9 Fig. 10

Fig. 11

In the digestive examinations, the color in connective tissues decreased remarkably following digestion by certain kinds digestives. From these experi- mental studies, the Mowry's method seemed to be superior to the Rinehart's for the 24 HIROSE, YASUTOMI, MURAI, YAMADA, KATAYAMA, TSUJINO, IWASA AND RAZZAQ

Fig. 12 Fig. 13 staining method of acid mucopolysaccharides. When the coloring procedure of the Prussian Blue was done in 70% acetone solution, the colors of both two methods were intensified. Even in the Rinehart's method, the color in connective tissues appeared when 70% acetone was used (Fig. 11), and the color in goblet cells was also more intensified. In the next step, the results of digestive examinations were investigated. After digestion by hyaluronidase, not only most of the positive part in connective tissues but also some positive parts in epitheliums disappeared. Fig. 12, the control slide, shows that acid mucopolysaccharides are colored well in the blood vessel wall of subserosa. Fig. 13 shows that after digestion by hyaluronidase the color disappeared completely except in the tunica intima of blood vessel. When sialidase was used, the positive substance in glands of fundus was digested perfectly, and when hyaluronidase was used, a stronger digestive reaction was also seen in the same section. DISCUSSION There are several methods to demonstrate iron of tissue, for example, a method using the organic reagent. Iron in vivoexists as a ferric type, therefore, the Prussian Blue reaction which was introduced to histochemistry by Pearls in 1866 for the first time is thought to be an excellent method. The Prussian Blue, however, is water- soluble, and therefore, it will be naturally washed away and diffuse in the tissue when distilled water is used as the solvent of the reagent. Therefore, it is natural to think that the color of the Prussian Blue should become light and sections should be stained diffusely. In order to improve these week points of his method this study was performed, in which we searched a method to decrease the solubility of the Prussian Blue as the first purpose. We thought about having the Prussian Blue reaction in the organic solvent. As the organic solvents, acetone and ethanol were chosen because (1) the reagents had to be dissolved well in the solvent, which was easily controlled by adding water to these solvents and (2) the solvent should not be ionized. If the solvent is ionized, there will be a possibility of chemical reaction between solvents and reagents. At the beginning, we studied the solubility of the Prussian Blue in acetone and ethanol solution. If the solubility does not decrease, we can not use them as solvents. MODIFIED METHOD OF PRUSSIAN BLUE REACTION 25

The solubility of the Prussian Blue in ethanol was almost same as in distilled water , even if the concentration of ethanol was high . Accordingly, ethanol was not suitable for the first purpose. The reason of high solubility in ethanol was probably the presence of OH-radical in it. If we use alcohols of higher molecular weight , we may be able to solve this problem , but they may be less water soluble. When we used acetone as the solvent, the solubility of the Prussian Blue decreased remark- ably. Accordingly, acetone was suitable for the first purpose . The reason may be that even acetone is water-soluble, it has not ionizing radical such as OH , which is in alcohol. The ionized HCI in the solution might have raised the solubility, but it was so slight that it was not a hazard for our first purpose . In the next place, we examined the solubility of the potassium ferrocyanide , the chief reagent, in the solvents. The potassium ferrocyanide was hardly soluble in 70% acetone, which was the lowest concentration in which the Prussian Blue was hardly soluble. But by adding HCI with 1% as final concentration , the solubility of potassium ferrocyanide increased more than four times. Therefore, the second problem was solved. It seemed that it was mainly due to the strong ionizing nature of HCI. But in more than 90% acetone, the solubility was very low, even if HCI was added. From these experiments in the test-tube, we came to the conclusion that the useful concentration of acetone was 70%-80%, in which the Prussian Blue was hardly soluble and potassium ferrocyanide, the chief reagent, was fairly well soluble. Before the experiment on the specimen, another one was performed upon spots of ferric chloride on filter papers. In this experiment we could obtain the perfect results in the 70%-80% acetone solution which was thought to be the most suitable concentration of acetone from the experiment of the test-tube. The minimum concentration of ferric chloride on spots which was able to demonstrate as color reaction in 70%-80% acetone solution was one fifth of that in distilled water. So we may conclude that the sensitivity of the color reaction in this solution was 5 times as sensitive as in distilled water. Moreover the diffusion on filter paper was scarcely seen in this solution. This good result was certainly caused by the fact that the Prussian Blue was hardly soluble and that potassium ferrocyanide, the chief reagent, was fairly soluble in acetone of such a concentration, which was supported by the experiment in the test-tube. In acetone solution of concentration less than 60%, the solubility of the Prussian Blue rose, therefore, it was thought that the Prussian Blue would be washed away considerably, the diffusion would occur and then the sensitivity would decrease. And in the acetone solution of concentration more than 90%, the color reaction would probably decrease because the solubility of potassium ferrocyanide decreased. In detecting iron in tissues, acetone solution also showed a remarkable results. The result in acetone of lower concentration was a little better than that in distilled water, but in 70%-80% acetone, the result obtained was as good as we expected, which was mainly because of low solubility of Prussian Blue and of fair solubility of potassium ferrocyanide. Clear reaction was seen in the stellated cells of Kupffer in the liver of dogs injected with 6MP, and the reaction was probably due to the hemosiderin which was produced in the spleen etc. and taken into the stellated cells. Prussian Blue in granular form was seen in liver cells especially those adjacent to bile canaliculi and also in bile canaliculi. This reaction was probably due to the iron 26 HIROSE, YASUTOMI, MURAI, YAMADA, KATAYAMA, TSUJINO, IWASA AND RAZZAQ which was in the process of being secreted into bile canaliculi. This fine reaction could not be demonstrated by the old method, because the Prussian Blue which was produced in the old method was washed away into the solvent and diffusion occurred resulting in staining of the part in which iron originally did not exist. In this way, we could obtain the very good result by using acetone of high concentration as the solvent in Prussian Blue reaction to demonstrate iron. We applied this method to the colloidal iron reaction of acid mucopolysaccharides and obtained very good result again. By comparing this method with the old method, we can explain the dif- ference of the color reactions according to the different kinds of colloidal iron used in the reactions. In the old method, at first, the Rinehart's method apparently seemed to be better than the Mowry's method because the former had good contrast. But when we used acetone of high concentration instead of distilled water in the procedure of coloring of iron, the reaction in connective tissues became stronger in the Rinehart's method, and the Rinehart's method became similar to the Mowry's method. Furthermore, both in the Rinehart's method and the Mowry's method, the coloring reactions in connective tissues were inhibited remarkably by hyaluron- idase and sialidase. Therefore, we could guess that the hyaluronic acid and the sialic acid, especially the former would be the main component of acid mucopoly- saccharides. We know that when we used distilled water as the solvent, we could scarcely catch these polysaccharides by the Rinehart's method. As above mentioned, the coloring reaction of iron in vivo and the colloidal iron reaction of acid mucopoly- saccharides were stronger when acetone was used than when distilled water was used. From the experimental result on filter paper, we could consider that the reaction in acetone was five times as sensitive as in distilled water.

SUMMARY

(1) Using 70%-80% acetone solution in the procedure of coloring reaction of the Prussian Blue, we could obtain the specimens which showed very sensitive and well localized reactions in detecting acid mucopolysaccharides. (2) The reasons of this good result are that the Prussian Blue produced is hardly soluble in 70%-80% acetone and that the potassium ferrocyanide, the chief reagent, is fairly well soluble in hydrochloric acid acetone solution. (3) According to the experiment on filter paper, the reaction in 70%-80% acetone is about five times as sensitive as that in distilled water and it is thought that we can demonstrate iron of minimum 0.005%. (4) When we used ethanol as the solvent, the result was as same as that in distilled water.

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