ACTA HISTOCHEM. CYTOCHEM. 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. SHUNTA HIROSE, MASAYUKI YASUTOMI, NOBUHIRO MURAI, RYOTARO YAMADA, ATSUSHI KATAYAMA, MOTOYOSHI TSUJINO, ZENJI IWASA and A.K.A. RAZZAQ. Department Surgery, Osaka University Medical School, Osaka. Received for Publication January 21, 1970. In Prussian Blue method for histochemical demonstration iron, some parts produced pigment are considerably washed away durg dyeg process, because pigment is water soluble. Consequently decrease and diffusion pigment occur and stag surroundg tissues take place. The color reaction was carried out 70% acetone solution order to decrease solubility pigment. Obtag sharp and clear demarcation, this method was applied successfully to colloidal iron reaction acid mucopolysaccharides and very strong and clear reaction was obtaed. In all histochemical methods to demonstrate iron, Prussian Blue reaction is best one. This method was described by Pearls 1866 for first time which iron is detected as Prussian Blue. This reaction combg with potassium ferrocyanide and Fe+++ is very sensitive, on or hand nearly all parts iron exist as a ferric type vivo. Therefore, Prussian Blue reaction is thought to be an excellent method demonstratg iron tissue. But because Prussian Blue produced reaction is water-soluble and distilled water is used as solvent reagents his method, Prussian Blue is easily washed away to solvent or to washg water. Therefore, it is difficult to prove small amount iron tissues, and Prussian Blue diffuses tissues and sharpness demarcation decreases. METHOD A) Solubility Prussian Blue. Four series 8 test-tubes were prepared. Two series m contaed, each, 10 ml ethanol solution diluted by distilled water 0, 30, 50, 60, 70, 80, 90, 100%; one series was added 0.1 ml HCI and or was not. In anor two series acetone was contaed stead ethanol. Then each test-tube 20 mg Prussian Blue was added and tubes were shaked and left for 3 hr at room temperature (20 Ž). The solubility Prussian Blue was thus vestigated. B) Solubility potassium ferrocyanide. Even if it was found that Prussian Blue was hardly soluble ethanol or acetone solution as result experiment A, it was thought that reaction would hardly occur, if potassium ferrocyanide, chief reagent reaction, was hardly soluble solvent. Therefore, it was necessary to vestigate 18
MODIFIED METHOD OF PRUSSIAN BLUE REACTION 19 solubility potassium ferrocyanide ethanol and acetone above mentioned concentrations. It was clarified after experiment A), its detail will be shown section result, that Prussian Blue was very soluble ethanol solution, but hardly soluble acetone high concentration. Therefore, 10% potassium ferrocyanide solution was dripped to distilled water and above described acetone solutions untill crystal potassium ferrocyanide was formed. The solubility was determed accordg to quantity potassium ferrocyanide solution used. In or series acetone solutions, which conc. HCI added up to 1% fally and solubility potassium ferrocyanide was also tested same manner. C) Color reaction on filter paper. If solubility Prussian Blue will be decreased acetone solution, solubility Prussian Blue specimens also will be expected to be decreased and good demarcation will be expected. In order to study slight difference solubilities produced by slight changes concentration solvent, color reaction was performed on spots Fe+++ on filter paper. For this purpose No. 51 filter paper For Chromatographic Analysis 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 spots on filter paper dried, y were immersed 1% hydrochloric acid acetone solution variable concentrations which were saturated with potassium ferrocyanide and left for 20 m at room temperature (20 Ž). The dex sensibility was determed by concentration ferric chloride spots which showed mimum color each concentration acetone solution, and dex diffusion, with size and density corona and sharpness spots. D) Detection iron tissues. We did this experiment order to know wher same results would beseen practical specimens. We used livers dogs jected with 6MP, fixed 10% formal, embedded paraff and cut at 6ƒÊ. And slides were immersed distilled water and acetone variable concentration. We did not use alcohol solution because Prussian Blue is very soluble alcohol. The reason usg such material is that it is easy for us to study reaction as liver tissue is homogenous and when jected with 6MP, hemolysis, liver cell disturbance and possession hemosider liver cell are seen. Countersta was performed by Feulgen reaction before stag iron. E) Detection mucopolysaccharides. The colorg procedure colloidal iron reaction acid mucopolysaccharides is same as Prussian Blue reaction which has been utilized to demonstrate naturally existent iron tissues. If Prussian Blue reaction will be improved, it can be applied to demonstrate acid mucopolysaccharides. In detectg iron tissues by method described above, we obtaed good results when reaction
HIROSE, 20 was done procedure will be high shown There method). water. because strated y conta formal more digestive deparaffizg reaction were stomachs colorg sharply cut material high examation slides were is, and what was is high concentration immersed a kd acid know One method used and as kd and countersta colloidal distilled iron were is demonwater, we sialidase was mucopoly- mucopolysaccharide than a materials They what is (Mowry's acetone were details mucopoly- iron. boilg hyaluronidase The resected to Its mucopolysaccharides. order acetone fished. acetone colloidal or diastase, 70% reaction acid In used surgically 6ƒÊ. concentration with were and kds iron producg compared at we TSUJINO, mucopolysaccharides. colloidal which several and acid procedure method) procedure before methods methods The 10% saccharide to Therefore, detectg result. different (Rehart's Both distilled fixed two acetone. iron section accordg method MURAI, YAMADA, KATAYAMA, IWASA AND RAZZAQ concentration colorg are saccharides dialysis YASUTOMI, done made after by Feulgen solution. RESULTS A) Solubility Prussian Blue. The Prussian Blue used experiment was completely dissolved distilled water as shown 1. So was it even 100% ethanol. The solubility decreased acetone solution. Though it was fairly well soluble 30% acetone solution, it was hardly soluble acetone solution higher concentration. In 50% acetone solution precipitation Prussian Blue creased remarkably, 70% Prussian Blue was almost soluble, and concentration higher than 80% it was completely soluble. ( 2) The Prussian Blue was dissolved better 1% hydrochloric acid acetone solution than acetone without HCI. But even existence HCl it was hardly soluble acetone solution concentration higher than 70%. B) 1 2 Solubility potassium ferrocyanide. The potassium ferrocyanide was also hardly soluble high concentration acetone. In 1% hydrochloric acid acetone solution, solubility potassium ferrocyanide creased remarkably. With HCI even 70% acetone solution, 1.3% potassium ferrocyanide was dissolved (Fig. 3).
MODIFIED METHOD OF PRUSSIAN BLUE REACTION 21 3 But when concentration acetone became higher than 90%, solubility potassium ferrocyanide decreased remarkably and only 0.3% potassium ferrocyanide was dissolved even with HCI. C) Color reaction on filter paper. Almost same reactions were observed distilled water and ethanol. No reaction was demonstrated m when concentration ferric chloride was under 0.025%. Even when concentration was over 0.025%, outle was not sharp and blue diffusion was seen. There was no difference between reactions distilled water and 1% hydrochloric acid acetone solution low concentration saturated with potassium ferrocyanide ( 4). When concentration acetone became high, reaction was clearly seen. In 70%-80% acetone solution, even 0.005% ferric chloride was demonstrated clearly, moreover outle was clear and diffusion was hardly observed. When concentration acetone was over 90%, reaction was hardly seen. D) Detection iron tissues. Experimental study was performed on dog livers treated with 6MP and excellent results were obtaed when 70 %-80 % acetone solution was used. We obtaed slightly better results 60% acetone than distilled water, but difference was not so remarkable. In 5 specimen was staed distilled water as usual. In 6 it was staed 75% acetone solution. In latter specimen, reaction was seen more clearly and demarcation was sharper than those distilled water. The Prussian Blue was seen spot-like sta scatterg liver lobules. Figures 7 and 8 show specimens high magnification,
22 HIROSE, YASUTOMI, MURAI, YAMADA, KATAYAMA, IWASA AND RAZZAQ 5 7 4 8 6 TSUJINO,
MODIFIED METHOD OF PRUSSIAN BLUE REACTION 23 which were staed distilled water and acetone. In specimen staed acetone solution, clear Prussian Blue reaction is seen not only stellated cells Kupffer, but also cytoplasm liver cells and bile canaliculi But specimen staed distilled water, such fe reactions are not seen. E) Detection mucopolysaccharides. Usg distilled water as solvent to sta iron bound acid mucopolysaccahrides, Rehart's method and Mowry's method were compared. In Rehart's method, Prussian Blue was seen maly goblet cells testal metaplasia and its contrast was clear (Fig. 9). But Mowry's method, fairly strong color was seen connective tissues besides metaplasia and color apparently seems to be due to deposit iron (Fig. 10). In remarkably mental 9 digestive examations, followg digestion studies, Mowry's by method 11 color connective certa kds digestives. seemed to be superior 10 tissues From decreased se to Rehart's experifor
24 HIROSE, YASUTOMI, MURAI, YAMADA, KATAYAMA, IWASA AND RAZZAQ 12 TSUJINO, 13 stag method acid mucopolysaccharides. When colorg procedure Prussian Blue was done 70% acetone solution, colors both two methods were tensified. Even Rehart's method, color connective tissues appeared when 70% acetone was used ( 11), and color goblet cells was also more tensified. In next step, results digestive examations were vestigated. After digestion by hyaluronidase, not only most positive part connective tissues but also some positive parts epiliums disappeared. 12, control slide, shows that acid mucopolysaccharides are colored well blood vessel wall subserosa. 13 shows that after digestion by hyaluronidase color disappeared completely except tunica tima blood vessel. When sialidase was used, positive substance glands fundus was digested perfectly, and when hyaluronidase was used, a stronger digestive reaction was also seen same section. DISCUSSION There are several methods to demonstrate iron tissue, for example, a method usg organic reagent. Iron vivoexists as a ferric type, refore, Prussian Blue reaction which was troduced to histochemistry by Pearls 1866 for first time is thought to be an excellent method. The Prussian Blue, however, is watersoluble, and refore, it will be naturally washed away and diffuse tissue when distilled water is used as solvent reagent. Therefore, it is natural to thk that color Prussian Blue should become light and sections should be staed diffusely. In order to improve se week pots his method this study was performed, which we searched a method to decrease solubility Prussian Blue as first purpose. We thought about havg Prussian Blue reaction organic solvent. As organic solvents, acetone and ethanol were chosen because (1) reagents had to be dissolved well solvent, which was easily controlled by addg water to se solvents and (2) solvent should not be ionized. If solvent is ionized, re will be a possibility chemical reaction between solvents and reagents. At begng, we studied solubility Prussian Blue acetone and ethanol solution. If solubility does not decrease, we can not use m as solvents.
MODIFIED METHOD OF PRUSSIAN BLUE REACTION 25 The solubility Prussian Blue ethanol was almost same as distilled water, even if concentration ethanol was high. Accordgly, ethanol was not suitable for first purpose. The reason high solubility ethanol was probably presence OH-radical it. If we use alcohols higher molecular weight, we may be able to solve this problem, but y may be less water soluble. When we used acetone as solvent, solubility Prussian Blue decreased remarkably. Accordgly, acetone was suitable for first purpose. The reason may be that even acetone is water-soluble, it has not ionizg radical such as OH, which is alcohol. The ionized HCI solution might have raised solubility, but it was so slight that it was not a hazard for our first purpose. In next place, we examed solubility potassium ferrocyanide, chief reagent, solvents. The potassium ferrocyanide was hardly soluble 70% acetone, which was lowest concentration which Prussian Blue was hardly soluble. But by addg HCI with 1% as fal concentration, solubility potassium ferrocyanide creased more than four times. Therefore, second problem was solved. It seemed that it was maly due to strong ionizg nature HCI. But more than 90% acetone, solubility was very low, even if HCI was added. From se experiments test-tube, we came to conclusion that useful concentration acetone was 70%-80%, which Prussian Blue was hardly soluble and potassium ferrocyanide, chief reagent, was fairly well soluble. Before experiment on specimen, anor one was performed upon spots ferric chloride on filter papers. In this experiment we could obta perfect results 70%-80% acetone solution which was thought to be most suitable concentration acetone from experiment test-tube. The mimum concentration ferric chloride on spots which was able to demonstrate as color reaction 70%-80% acetone solution was one fifth that distilled water. So we may conclude that sensitivity color reaction this solution was 5 times as sensitive as distilled water. Moreover diffusion on filter paper was scarcely seen this solution. This good result was certaly caused by fact that Prussian Blue was hardly soluble and that potassium ferrocyanide, chief reagent, was fairly soluble acetone such a concentration, which was supported by experiment test-tube. In acetone solution concentration less than 60%, solubility Prussian Blue rose, refore, it was thought that Prussian Blue would be washed away considerably, diffusion would occur and n sensitivity would decrease. And acetone solution concentration more than 90%, color reaction would probably decrease because solubility potassium ferrocyanide decreased. In detectg iron tissues, acetone solution also showed a remarkable results. The result acetone lower concentration was a little better than that distilled water, but 70%-80% acetone, result obtaed was as good as we expected, which was maly because low solubility Prussian Blue and fair solubility potassium ferrocyanide. Clear reaction was seen stellated cells Kupffer liver dogs jected with 6MP, and reaction was probably due to hemosider which was produced spleen etc. and taken to stellated cells. Prussian Blue granular form was seen liver cells especially those adjacent to bile canaliculi and also bile canaliculi. This reaction was probably due to iron
26 HIROSE, YASUTOMI, MURAI, YAMADA, KATAYAMA, TSUJINO, IWASA AND RAZZAQ which was process beg secreted to bile canaliculi. This fe reaction could not be demonstrated by old method, because Prussian Blue which was produced old method was washed away to solvent and diffusion occurred resultg stag part which iron origally did not exist. In this way, we could obta very good result by usg acetone high concentration as solvent Prussian Blue reaction to demonstrate iron. We applied this method to colloidal iron reaction acid mucopolysaccharides and obtaed very good result aga. By comparg this method with old method, we can expla difference color reactions accordg to different kds colloidal iron used reactions. In old method, at first, Rehart's method apparently seemed to be better than Mowry's method because former had good contrast. But when we used acetone high concentration stead distilled water procedure colorg iron, reaction connective tissues became stronger Rehart's method, and Rehart's method became similar to Mowry's method. Furrmore, both Rehart's method and Mowry's method, colorg reactions connective tissues were hibited remarkably by hyaluronidase and sialidase. Therefore, we could guess that hyaluronic acid and sialic acid, especially former would be ma component acid mucopolysaccharides. We know that when we used distilled water as solvent, we could scarcely catch se polysaccharides by Rehart's method. As above mentioned, colorg reaction iron vivo and colloidal iron reaction acid mucopolysaccharides were stronger when acetone was used than when distilled water was used. From experimental result on filter paper, we could consider that reaction acetone was five times as sensitive as distilled water. SUMMARY (1) Usg 70%-80% acetone solution procedure colorg reaction Prussian Blue, we could obta specimens which showed very sensitive and well localized reactions detectg acid mucopolysaccharides. (2) The reasons this good result are that Prussian Blue produced is hardly soluble 70%-80% acetone and that potassium ferrocyanide, chief reagent, is fairly well soluble hydrochloric acid acetone solution. (3) Accordg to experiment on filter paper, reaction 70%-80% acetone is about five times as sensitive as that distilled water and it is thought that we can demonstrate iron mimum 0.005%. (4) When we used ethanol as solvent, result was as same as that distilled water. REFERENCES 1. Arcadi, J.A.: Some polysaccharide components prostate gland dog. Anat. Rec. 112; 593, 1952. 2. Constante, V.S., Mowry, R.W.: Histochemical demonstration sialomuc human eccre sweat glands. J. Inv. Dermatology 46; 536, 1966. 3. Hale, C.W.: Histochemical demonstration acid polysaccharides animal tissues. Nature 157; 802, 1946.
MODIFIED METHOD OF PRUSSIAN BLUE REACTION 27 4. Lillie, R.D., Emmart, E.W. and Laskey, A.M.: Chondromucase from bove testis and chondromuc umbilical cord. Arch. Path. 52; 363, 1951. 5. Mowry, R.W.: Improved procedure for stag acid polysaccharides by Miiller's colloidal (hydrous) ferric oxide and its combation with Feulgen and Periodic acid-schiff reactions. Lab. Invest. 7; 566, 1958. 6. Pearls, M.: Nachweis von Eisenoxyd gewissen Pigmenten. Virchows Arch. 39; 42, 1867. 7. Qutarelli, G.T., Suiki, S., Hashimoto, Y. and Pigman, W.: Studies sialic acid contag mucs bove submaxillary and rat sublgual glands. J. Histochem. Cytochem. 9; 176, 1961. 8. Rehart, J.F. and Abul-Haj, S.K.: An improved method for histologic demonstration acid mucopolysaccharides tissues. Arch. Path. 52; 189, 1951.