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A Modified Method of the Prussian Blue Reaction For ACTA HISTOCHEM. CYTOCHEM. Vol. 3, No. 1, 1970 A MODIFIED METHOD OF THE PRUSSIAN BLUE REACTION FOR THE HISTOCHEMICAL DEMONSTRATION OF IRON, 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 solubility 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 potassium ferrocyanide 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 ethanol 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 solubilities 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.
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