The histochemical methods for calcium and inorganic phosphate, e. g. alizarin red S methods (McGee-Russell and Dahl), silver nitrate methods (von Kossa and Heller-Steinberg) and molybdate method (Serra-Feigl) are studied in a model experiment and in the undecalcified sections of bone and dentin.
The Dahl's method is superior to the McGee-Russell's in respect of its specificity. Washing the slides in water for 3 minutes is found to be the most suitable method for differentiation. Among the silver nitrate methods for calcium and calcium deposits in the calcified tissues, especially in dentin, the Heller-Steinberg's method seemed to be more specific and sensitive for inorganic phophates than the von Kossa's. Investigating the possible effects of decalcifying solutions on histochemical staining reaction of various inorganic salts in the model experiment, an important suggestion is given in explanation of the destructive mechanism of inorganic components in dental caries.
Acknowledgement Grateful acknowledgements are made to Prof. Dr. Taro Terasaki for his constant encouragement and valuable advice throughout this work.

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The author found some new excellent cyanine dyes to demonstrate the metachromatic property of tissue elements. With these dyes, distribution of metachromatic elements in rat tissue was traced. The staining pattern of tissue elements with cyanine dyes after sulphation technique was observed. The results were compared with PAS staining reactions.
This new histochemical method for metachromatic staining will be useful for study of substances that can be sulphated in tissue sections.

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³H-uridine and ³H-thymidine autoradiography of polytene chromosome revealed that the bands are inactive in RNA synthesis and late replicating in DNA synthesis. These characteristics are common in inactivated DNA portions in the insect and mammalian chromosomes. The distribution of the inactivated portions on the chromosome defines irreversible cytodifferentiation of the cell. This type of inactivation can only appear in the metazoan chromosome which is composed of many autocatalytic subunits. The organization of bacterial chromosome is incompatible with this type of multiple inactivation, thus impossible to manifest cytodifferentiation.
This investigation was supported in part by a PHS research grant GM-09469-02 from the Division of General Medical Sciences U. S. Public Health Service.

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A combination of Adams' DMAB-nitrite reaction, counterstaining of nuclei with Schiff's reagent and routine PAS reaction is described. The whole procedure of this combination stains tryptophane, DNA and polysaccharides in single sections in different colors. The effects of various fixatives on the DMAB-nitrite reaction is also tested.

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The antigenic preservation of various fixation procedures as applied to human placenta was re-examined by means of hemagglutination inhibition test. The effect of deoxycholate and sonic vibration on its antigenicity was also examined.
The freeze-substituted sections, cold acetone fixed ones and frozen ones are all suitable for fluorescent antibody technique, but not the formalin fixed ones. Not all of antigenic substances, however, may be detected in these sections by this method.

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The methods were evaluated to obtain large volumes of labeled proteins with bright specific and negligible nonspecific staining by means of DEAE-cellulose column chromatography and analytical techniques. Conditions to prepare conjugates to yield fruitful result were coupling ratio of F.I.T.C. and protein 1: 125 at 5°C pH 9.6 and coupling ratio of 1: 250 in widely used condition, at 5°C pH 9.0.

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New FSM devised by ourselves was described. This method gave excellent observations in the morphological and histochemical examinations.

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In the freeze-substitution method for histochemical preparation of the tissue section, an alternative technique was introduced by Lison and later improved by Feder & Sidman. It was based on adding some fixatives to the substitution medium in which tissue blocks were dehydrated at lower temperature. Some modifications of this method and their results have been published by us also (Sugimoto & Yagi). The present paper is concerned with further investigations on other modifications suitable for fixation of mucopolysaccharides or mucoproteins and on their application to the secrtory granules of salivary and some other glandular tissues in rats.

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By histochemical studies on monoamine oxidase (MAO), we demonstrated some new findings about MAO activity in the frog retina under the photopic and scotopic conditions.

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Diphosphopyridine nucleotide diaphorase, succinic dehydrogenase, and glucose-6-phosphate dehydrogenase were studied on the interfascicular neuroglia and the nerve fiber of the optic nerve after enucleating the eye ball.
1. The dehydrogenase activity of the glial cell was generally stronger than that of nerve fiber with all substrates.
2. Increase of dehydrogenase activity was not found on the one week after enucleation.
3. The dehydrogenase reaction showed the greatest increase at two weeks. The morphological changes of interfascicular neuroglia (oligodendrocytes) consisted in the enlargement of the cells and delicate ramification of their processes.
4. The localization of enzymes in the glial cells was homogeneous throughout the cell bodies and their procceses in enucleated rats. In the guinea pigs hypertrophic interfascicular neuroglia (oligodendrocytes) were of slender or irregular shape and their processes were thick compared to the large oval or triangular shape in the rabbits. The distribution of enzyme activity outlined by the strong reaction appeared as granules in the perinuclear cytoplasm.
5. In the rats on the fourth week after the injury, large cells (perhaps spongioblast) gave an intense reaction with homogeneous formazan precipitates.
6. The large cells and surrounding interfascicular neuroglia (oligodendrocytes) contained strongly black positive granules of formazan in the degenerating nerve on the eighth week after injury. The response of the glial cells showed slight increase in all enzyme reactions.

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1) No enzyme activities for succinic dehydrogenase and DPN-diaphorase in myelin sheath of normal, degenerating and regenerating sciatic nerve could be demonstrated by histochemical methods. Non-specific esterase activity could be found in the nerve fiber but not in axons, their distribution in nerve fiber was in irregular pattern with fine positive granules.
2) The myelin structure in fresh frozen sections showed circular thin threads, and this structure coincided with electron microscopic view.
3) Alteration of succinic dehydrogenase and DPN-diaphorase in the proximal stump following sciatic neurotomy: Axonal swelling accompanied with a damming of succinic dehydrogenase and DPN-diaphorase at the end site of axon and increased enzyme activity in the Schwann cell were noted.
4) The changes in monoamine oxidase activity in the regenerating nerve fiber in proximal stump showed a very interesting localization, during the regeneration of sciatic nerve, the mid-portion of proximal stump demonstrated strong monoamine oxidase activity in the outer surface of the axon and this may indicate the axon-Schwann cell membrane.

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Higher levels of the succinic dehydrogenase, DPN-diaphorase and cytochrome oxidase activities at the cell membrane of spinal motor neuron, motor neuron in reticular formation, Purkinje cell in the cerebellar cortex and large pyramidal cell in the cerebral cortex of mouse, rat, guinea pig and rabbit have been described.
The distribution patterns of succinic dehydrogenase and DPN-diaphorase were shown as spotted in type and cytochrome oxidase arranged as fine granular in type.
Although the physiological significance of these enzymes and their histochemical investigation by means of electron microscope on the synapse is still unknown, a suggestion is made that the oxidative enzymes probably are related to synaptic mitochondria and thus they play a part of the energy changes concerned with impulse transmission from neuron to other neuron in the central nervous system.

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The recent development of enzymocytochemistry encourages further investigation of nerve cells in non-physiological states, applying reliable staining methods of several kinds of enzymes.
This experiment aimed to observe the changes of enzymatic activities of chromatolytic motor nerve cells in anterior horn of rabbit spinal cord, in which the retrograde reaction was induced operatively.
Observations upon the nerve cells in simply induced chromatolytic state, with reasonable staining controls of normal cells in symmetrical anterior horn, may allow to discuss some possible metabolic conditions of nerve cells in this state.

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PhR deficiency in skeletal muscle fibers caused by sympathetic ganglionectomy was demonstrated histochemically in infant dogs. Slight decrease of SDH activity and almost intact activity of ChE in motor end-plate were also observed. The relation between PhR deficiency and sympathetic ganglionectomy was discussed.

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During metamorphosis striking changes occur in various organs and tissues of amphibia. Over the years many studies have been carried out and several theories were presented to explain the mechanisms of metamorphosis. One of the present authors has studied connective tissue of the skin during metamorphosis (Niizima, 1955) and the behavior of metamorphosing tissues in tissue culture (Niizima, 1956). In our laboratory, electoron microscopic study of amphibian tissues during metamorphosis is also in progress.
Most of the investigations which have been reported up to the present were restricted to histological or experimental studies. Although there have recently been many biochemical studies, the authors have been unable to find any reports of histochemical investigations in this field so far.
Since histochemistry shows the distribution of substances in tissue but cannot accurately determine their exact quantities, it becomes necessary, for the understanding of the phenomena in metamorphosis, to compare biochemical data with histochemical findings.
In the present investigation, attempts were made to determine whether any changes in the localization of activities of some enzymes in the metamorphosing tail tissue could be detected, in order to clarify which enzymes play an important role in the metamorphosis or involution of the tail.
The tail was chosen as the first object of our studies since the retraction of the frog tail is one of the most conspicious phenomena among the many changes occuring during metamorphosis. The enzymes investigated were alkaline and acid phosphatases, leucine aminopeptidase, succinate dehydrogenase and cytochrome oxidase.

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The distribution patterns of oxidative enzymes during wound healing were demonstrated histochemically by the Nitro BT method. Significant alterations in histochemical localization of oxidative enzymes participating the several metabolic pathways were observed in epithelial and mesenchymal structures of the regenerating stage.

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Demonstration was made of the localization of various hydrolytic and oxidative enzymes in the rat's callus, which was formed two weeks after a long bone fracture. With the following results: All the hydrolytic and oxidative enzyme reactions examined were observed markedly in proliferating and hypertrophic cartilage cells, and moderately in the matrix of fibrous callus and the surface of trabecu lea in osserous callus. No enzymatic reactions were seen in the matrix of cartilaginous callus, with the exception of alkaline and acid phosphatase and glycosidase. Some of the enzymes studied showed their own characteristic localization, that is, alkaline phosphatase reaction was marked in the matrix of fibrous and cartilaginous callus, acid phosphatase reaction was marked in the osseous callus, glycosidase reaction was also marked in cartilage cells, succinic dehydrogenase reaction was marked in the transitional portion of fibrous callus to cartilaginous callus and osteoclasts, malic, lactic and glucose-6-phosphate dehydrogenase reactions were also marked in the fibrous callus and cartilage cells. In general, the reaction of DPN dependent dehydrogenases in proliferating cartilage cells was stronger than that of TPN dependent dehydrogenases.

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1) All the four enzymes stained and fat staining showed marked differences between 1 1/2 and 2 1/2hrs. of occlusion.
2) Both ischemic and hydronephrotic kidneys revealed dilated tubules. Some of them showed low emzymatic activity, while the others remained to possess normal enzymatic activity. The latters are considered to be hypertrophied to compensate necrotic or low functioning tubules.
3) The significance of increased I.R. and S.D. activities in the ischemic kidneys is not known.
4) Marked differences were observed in the enzymatic activities between hydronephrses produced by ureteral ligation of less than 10 days and longer than 20 days. This fact may contribute to know whether or not renal tissue is viable.
5) The fact that S.D. and TPN activities increased in the medulla and decreased in the cortex is interesting, when the experiment with Na²⁴ showing higher concentration in the medulla is referred.
6) No changes of enzymatic activities were seen in the contralateral kidneys in hydronephrotic and ischemic cases. This means that the healthy kidneys contain a certain amount of enzymes enough to compensate the decreased function of the treated kidneys. Renal functional reserve is accompanied by renal enzymatic reserve.
7) Fatty degeneration was observed in slightly dilated tubules hydronephrotic and ischemic kidneys. It suggests that fatty changes occurred at the beginning of cellular degenration.

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Pyridoxal phosphate decreased in intensity of the histochemical reaction for phosphorylase in tissue sections. It was difficult to prove histochemically that a combination of pyridoxal phosphate with apophosphorylase might be demonstrated through the phosphorylase reaction. It was assumed that the effect of pyridoxal phosphate upon apophosphorylase and upon phosphorylase itself is different respectively.

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The phosphorylase activity was histochemically demonstrated surely in certain tissue cells by the iodine reaction procedure incubating in the Mölbert's substrate mixture for electron-microscpic observation, but it was only roughly demonstrable by the lead phosphate technique. The erratic reaction which may occur in the metal-salt procedure was unavoidable.

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A histochemical distribution of three dehydrogenase systems, succinicdehydrogenase, DPN-dependent and TPN-dependent dehydrogenases in majorsalivary glands, liver, testis, kidney and spleen of adult male mice exposedto a single whole-body γ-ray (1000γ) was observed. The histochemical significance in the changes of stainability was detected in succinic, α-glycerophosphate, glucose-6-phosphate and isocitric dehydrogenase of major salivaryglands, liver and testis.
The above four dehydrogenases in gland-tissues generally showed a gradualdecrease and the lowest reactions from the 2 nd day to the 4 th day after theirradiation. Among the rest, a decrease of α-glycerophosphate and glucose-6-phosphate dehydrogenase in the seminal epithelium of the testis was mostconspicuous. From the 7 th day to the 10 th day after the irradiation, thesurvived duct epithelia of salivary glands maintained a positive reaction ofsuccinic, glucose-6-phosphate and isocitric dehydrogenases. The above decreaseof stainability in oxidative enzymes affected by the irradiation was occasionallyassociated with morphological destructions, indicating a suggestion of indirecteffects of γ-irradiation.

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The Nadi reaction has been known as the histochemical technique for the demonstration of cytochrome oxidase. But this method has several shortcomings as following ; diffusion and crystallization of the pigment in the tissue sections, rapid fading and lipid solubility of the pigment, and difficulty for counterstaining of the nucleus due to the instability of the pigment by the procedure.
Burstone roported his works in which he employed, for histochemical demonstration of cytochrome oxidase, many sorts of combinations of naphthols and amines, or amines alone, in addition, a series of non naphtholic compounds with reactive methylen groups.
Independently of his works, we tried to obtain the stable red dye stuff which might be adequate for the counterstain. The 1-phenyl-3-methyl-5-pyrazolone and diethyl-p-phenylendiamine were employed in stead of alpha-naphthol and dimethyl-p-phenylenediamine. The employment was found to be useful for the demonstration of this enzyme and superior to the original Nadi method. The counterstaining of the nucleus using methylgreen gave good contrast.

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Animals used were Urolonchae domesticae, and sections employed were 10μ paraffin sections of the liver. 1) The effect of temperature of water on acid and alkaline GMP-ases, TDP-ases and ATP-ases were different. Acid GMP-ase was inactivated at 70°C, acid TDP-ase at 80°C and acid ATP-ase at 90°C. Alkaline TDP-ase and alkaline ATP-ase were inactivated at 50°C, and alkaline GMP-ase at 60°C. As a result, a GMP-ase- and TDP-ase-inactivated acid ATP-ase preparation was obtained by the 80°C water pretratment. However, an immersion of sections in 80°C water for 30 minutes caused a fuzzy appearance. 2) The effects of M/10 acetic acid, M/10 sodium barbiturate and distilled water at 0°C on acid and alkaline GMP-ases, TDP-ases and ATP-ase were different. Sodium barbiturate markedly inactivated acid GMP-ase and completely acid TDP-ase, but exerted almost no effects on acid ATP-ase. Accordingly, the sodium barbiturate-treated ATP-ase preparation showed a distinct localization of ATP-ase activity at pH 5.0, namely in the mitochondria. Acid TDP-ase was markedly inactivated also by immersing sections in distilled water for 18 hours in a refrigerator, showing the properties of lyoenzymes. With this and the diffuse intracellular localization of the activity, the localization of acid TDP-ase was indicated to be in the microsome.

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Peptidase is, as is well known, a generic term of enzyme series which split the linkage of peptic bonding. In this group, carboxypeptidase, amino-peptiase, dipeptinase, prolinase and prolidase are classified. For histochemical study on the substrate specificity of peptidases still more new method needed to be developed. At the second general meeting of the association, we reported a new method, regarding capture of naphthylamine from naphthylaminic substrate of soluble salts. Insoluble compounds are not available as a substrate of enzyme in this method.
We synthesied palmityl-alpha-naphthylamine, palmityl-beta-naphthyl-amine and palmityl-anilide. These substances are not soluble in water. We can make a relatively stable emulsion of the substances by use of non-ionic emulsifier as we described in the following mehtod.
In the new method, released palmitic acid was precipitated by calcium ion as is in the method of lipase histochemistry. The calcium was replaced by lead in a lead nitrate solution as is in the Gomori method for lipase. The lead was visualized by staining with Alizarin Red according to the method of Takamatsu, Ozawa and Izaki.

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1. Pig pancreas extract is examined by means of paper chromatographic technic using 1.5% NaCl and 10% acetone buffer solution pH 5.0 as a developer. Esterase and lipase are separated and they showed color reactions in different places respectively. At least two spots are found in esterase reaction.
2. Eleven kinds of effectors are applied to the separated esterase portion on paper chromatogram and resulting color reactions are examined. At least in two spots, different color reactions are noted, depending on the different substances applied Atoxyl, Eserine and Thyroxine showed strong color reaction in the spot of relative larger Rf value. Fluoride, Urethane, Qunine, Cocaine and Nitromin showed weak color reaction (inhibition) in the spot of smaller Rf value. Strychinine and 1-Leucylglycylglycine showed inhibition in both spots in same degree. Taurocholate showed activation in both spots.

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The esterase activity was histochemically demonstrated in free cancer cells of Yoshida hepatoma, AH 13 and AH 39 by the azo dye method using β-naphthyl acetate and naphthol-AS acetate as the substrate and also by the metal salt technique using Tween substrates. The activity remains localized in the intracellular specific structures suggesting the centriosomes and the Golgi area in the hepatoma cells.

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Since 1914, an enzyme which catalyzes the dehydrogenic oxidation of D-sorbitol has been demonstated by means of biochemical methods. This enzyme was named “sorbitol dehydrogenase”.
This paper deals with a histochemical study of sorbitol dehydrogenase activity.

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Since the development of the Tween method for lipase and esterase by Gomori, these enzymes have been intensely studied histochemically. Up to date, however, no histochemical studies were performed on the cholesterol esterase activity in animal tissues. The enzyme, which could hydrolyze the fatty acid esters of cholesterol, was prepared from animal tissues and the name “cholesterol esterase” has deen applied. Byron et al (1953) and Hernandez and Chaikoff (1957) have studied this enzyme biochemically.
From the histochemical points of view, the problems of the substrate specificity of esterases, particularly those of lipase and non-specific esterase, are very interesting and also of grest importance. Therefore, it is expected that the histochemical findings of the cholesterol esterase activity may make some contributions to the solution of the problems of substrate specificity of esterases.

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It has been proved that the chief component of the iron hematoxylin stained granules found in rat adrenocortical cells as well as acidophils of rat anterior pituitary may be considered to be protein bound phospholipid.
Further conclusion has been obtained that the increased iron hematoxylin stained granules should be regarded as the bodies intimately concerned in the corticoid formation.

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Morphological and chemocytological studies have been made of three types of protein granules visualized in the parathyroid cells of the Japanese quail (Coturnix coturnix japonica). These granules are reactive for coupled tetrazonium, DDD diazo blue B and HNAH diazo blue B stains respectively. On the grounds of the morphological and chemocytological properties of the protein granules, glycogen and ribonucleic acid in the parathyroid cells, the cytophysiological significances of the granules have been discussed.

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Six enzymes of the intestinal metaplasia were investigated histochemically.
Though leucine aminopeptidase activity was not found in normal gastric mucosa, marked reaction was detected in the intestinal metaplasia. The other enzymatic activity was also increased in it.
Intestinal metaplasia and small bowel were identical morphologically and histochemically. However, the pathogenesis of intestinal metaplasia remains to be solved.

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Histochemical studies of the gastric cancer cells showed the following results:
1. Alkaline phosphatase, leucine aminopeptidase and ATP ase activity was not found or slight in the gastric cancer cells.
2. Though activity of succinic dehydrogenase and glucose-6-phoshatase was increased in the differentiated cancer cells, it was decreased in the undifferentiated cancer cells.
3. NADH dehydrogenase and, β-glucuronidase revealed much intense activity than the other enzymes. Moreover, activity of these enzymes varied corresponding to the degree of differentiation of the gastric cancer; undifferentiated cancer cells and infiltrating solid carcinoma without distinct glandular structures showed much intense activity than differentiated cancer cells.

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The acridine orange (AO) fluorescence method for the diagnosis of cancer, developed by L. von Bertalanffy, utilizes increased amounts of cytoplasmic ribonucleic acid in malignant cells to demonstrate them in brilliant reddish orange colors. Many authors have studied the method for the cytodiagnosis of cancer with materials from the female genital truct, gastric washings, and sputurn. Coriphosphine O (CO) staining has been used conventionally for the use of RNA marker on tissue culture cells, frozen sections, and other specimens. However no practical works have been reported for the cytodiagnosis of cancer with CO staining. In this paper we studied the representation of PNA in malignant cells to test the diagnostic reliability of CO staining, comparing with those of stained independently by AO fluorescence method on different smears from the same specimens.

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