For the histochemical demonstration of alkaline phosphatase, various methods such as azo dye method and cobalt calcium method have been employed. It has been difficult to check the precise localization of the enzyme on the tissue sections, because these reactions have been usually accompained with adsorption or diffusion of the reagents and the reaction products on tissues.
We found a modified method by adding the high concentration of sodium chloride solution into the reagents mixture so that the precise localization could be obtained with minor diffusion and adsorption of the reagents and the products on the tissues.
The mechanism of protection against adsorption and diffusion of the reagents and the products seemed to be mainly due to the salting-out effect by the sodium chloride. But it was not decided yet whether this phenomenon was truly due to the protection against adsorption and diffusion of the reagents and the products or was due to the positional variation of the disturbance of the enzyme activity by sodium chloride in tissues or cells. We have therefore biochemically and histochemically tried to explain the mechanism of the salt reaction on the activity of alkaline phosphatase.

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It was paper chromatographically found that the enzymatic ATP-A5′ MPransphosphorylation (the forward reaction in Equation (1) ) with crude extract of myokinase of rat kidney tissue leads to the production of ATP and A5′ MP in the incubation time as 15, 30 minutes, 1 hour and 2 hours. The evidence on the activity of myokinase contained in rat kidney tissues was supported by the fact that ADP was enzymatically formed in the incubation period ranging from 15 minutes to 2 hours when ATP and A5′ MP were used as substrate at pH 9.5.
The enzymatic activity of crude myokinase preparation toward ATP and A5′ MP was evidently confirmed by means of electrophoretic technique.
Thus, from the results obtained it was concluded that the enzymatic activity of myokinase plays an important rôle in the production of A5′ MP from ADP which formed in the incubation mixture under the action of ATPase. Accordingly, the fact seems to be much important to the differentiation and identification of true ATPase in tissue section.

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The histochemical demonstration of UDPG-pyrophosphorylase may be possible by use of polysaccharide synthesis from glucose-1-phosphate with uridine triphosphate by the enzyme activity under the suitable conditions combining pH, co-factor, activators and inhibitors, in spite of the difficult differentiation from amylophosphorylase. However, the distinct differentiation between two pathways still remains in difficulty.

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For histochemical demonstration of sulfatse activity of tissue sections, some experiments were tried according to our new principles. The methods and results were reported. In compare with other methods for the enzymes, these new methods seemed to be not so excellent at present time, but the authors believe that the new principle will cause great contribution in enzyme histochemistry.

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A new histochemical method for lipase was developed using naphthol-AS nonanoate as a substrate and sodium taurocholate as an activator.
In a comparison with esterase, pancreatic-like lipase activity was studied in various tissues. As a result, it was disclosed that the pancreatic acinar cells show the highest activation with sodium taurocholate, and lipase activity is located in the region immediately surrounding the zymogen granules and not within the granules themselves.
Maximum activity of esterase and lipase differed among various tissues of the rat. Esterase activity is most intense in the liver and kidney followed by pancreas. Pancreatic-like lipase activity is most striking in pancreas, less so in the liver and, in the kidney the activity is rather weak.
Because of the exceptional esterase activity of macrophages, mast cells and pericytes of the cerebral capillaries, pancreatic-like lipase activity should be ascribed to them with caution and only in terms of the difference between reaction without and with sodium taurocholate.

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The activities of some oxidative enzymes, which were markedly decreased or completely lost by dewaxing processes, were restored by the incorporation of phospholipids, Co Q₁₀ or β-lipoprotein into the incubation media.
Oxidative enzymes were divided into five groups on the basis of the grades of recovery of enzyme activities by phospholipids, Co Q₁₀ or β-liporotein.

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A methodological investigation and an application were presented on histochemical procedures for adrenal medullary catecholamines with new potassium dichromate technique. Wood's method for light microscopy and Wood-Barrnett's method for electron microscopy were employed. Combination of these procedures was also tried. Changes of adrenal norepinephrine and epinephrine were observed after reserpine, insulin and α-methyldopa administration. Characteristics of these method were discussed, compared with Hillarp-Hockfelt's method.

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Histochemical test for Zinc was first reported by Okamoto and his coworkers in 1944 with use of diphenylthiocarbazide and dithizone. Since then a number of other reports of various methods came in attention but all of these were based on the identical principle. Mager's method is one emphasis placed on a specificity for Zinc only when freshly frozen section. was made of the Islets of Langerhans of pancreatic tissue, but admitted negative result almost always if alcohol fixation of the tissue would be followed by sectioning in paraffin embedding.
These findings have been reportedly true with the results obtained by applying the method of Amann and Wolff to the fresh frozen section. A positive test, however, can ce obtained by Okamoto et al or Maeda et al method using alcohol fixed, paraffin section. Timm, also, had succeeded to obtain positive test even with alcohol fixation and paraffin sectioning with application of magnesium dithizonate.
Okamoto at the same time stated that the reaction for Zinc was hardly obtainable if ammonium chloride or ammonium carbonate solutions was employed as a medium in differentiating the other metals which would present reaction resembling Zinc.
In our investigation, nevertheless, we have reached a conclusion that it is always possible to obtain a positive test when freshly frozen section were tried. Present experiment was carried out to seek possible explanation if any for the nature of difference in reaction between fresh section specimen and paraffin section one after alcohol fixation.

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A method of fixing-decalcification (simultaneous) of hard tissues was studied from a histochemical poit of view, and 10% DETA-10% formalin solution was found to be recommended for this purpose. Histochemical observations on alkaline and acid phosphatases, non-specific esterase, polysaccharides, and proteins in normal rat incisors and femurs treated with the fixing-decalcification method were reported with brief discussion.

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There may be a pool of glycogen metabolism in tumor cells. The phosphorylase activity is always demonstrable in tumor cells, even when glycogen deposits are not observed. In AH13 richer glycogen, it tends to decrease and to disappear during DNA-synthesis and mitotic division of tumor cells, while just then phosphorylase is activated. The detailed data will be reported in future.

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Enzymatic activities and distributions of NADP-dependent glucose-6-phos-phate and 6-phosphogluconate dehydrogenases which act as key enzymes for the pentose phosphate pathway were histochemically detected in 82 cases of human benign and malignant tumors. Although there were considerable vari-ations in both enzymatic activities in terms of the origin of tumor, glucose-6-phosphate dehydrogenase generally had a high activity in tumor cells, while6-phosphogluconate dehydrogenase showed rather a low activity.
It is suggested that the NADP produced by the action of these dehydro-genases seems to be due to various synthetizing mechanism in organs, e. g., insulin production in pancreas, hydroxylation of steroid synthetizing processess in adrenal glands and in the ovary. The above findings seem to reflect one of the factors regulating enzymatic changes in tumors. Further studies on this aspect are under way.

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Recent advances in enzyme histochemistry have introduced very valuable methods to estimate the specific and non-specific metabolic activities of tissue components in close relationship with their pathohistological composition. Studies on human neoplastic tissues with histochemical observation of various enzymes have been made by several workers in recent years, but only a few reports seem to have been made on a systemic comparative study of the benign and malignant neoplastic tissue of human breast.
In the present study enzymatic activities were investigated through the histochemical method in human breast tissue, mainly carcinoma, mastopathy, fibroadenoma, etc. Enzymatic activities were compared between benign and malignant lesion, cancerous and non-involved part, cancer perfused with anti. cancer agents and non-perfused cases, and among various histological types of cancer, and then the experiment in which frozen section of breast cancerous tissue was contacted with anticancer agents in vitro, was done in the purpose of justifying the effect of anticancer agents.

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1. The human gastric mucosa and small intestine taken by biopsy were investigated histochemically.
2. The gastro-intestinal tract of the normal experimental animals (rabbits, rats and dogs) was also investigated histochemically.
3. Fifteen enzyme activities such as SDH, DPN diaphorase, DPN dependent dehydrogenases, phosphatases etc. were investigated.
4. Intestinal metaplastic cells were identical with the small intestinal mucous cells morphologically and histochemically.
5. These results were shown in Table 1. 2 and 3.

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In the skin, as in other organs, metabolic pathways such as Embden-Myerhof pathway, Warburg-Dickens pathway, Eisenberg pathway and TCA cycle are present, and energy obtabined through these way is untilized for keratinization, secretion of sweat and sebum and mitotic activity in epidermis. In this report some enzyme activities in the course of energy metabolism in human embryonal skin was observed histochemically, and comparison to those of adult skin was made.

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Auf die vielerörterte Frage nach der funktionellen Bedeutung der Epiphyse läßt sich bisher keine auch nur annähernd befriedigende Antwort erteilen. Zwar wird das rätselhafte Organ in die Reihe der inkretorischen Drüsen eingegliedert, doch rechtfertigen weder histologische noch physiologische Befunde diese Eingruppierung vollkommend. In der vorliegenden Studie wird die Epiphyse von einigen Säugetieren histochemisch sowie elektronenmikroskopisch im Hinblick auf ihre sekretorische Funktion untersucht.

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A ceroid-like pigment is observed in the Kupffer's cell and histiocytes of mouse liver following subcutaneous administration of sodium silicate, sodium silicate plus prednisolone and sodium nucleate plus prednisolone. Comparative histochemical studies are made between this ceroid-like pigment and the ceroid pigment experimentally produced in mice of the same strain which have been fed on a vitamin E deficient diet.

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Proliferated bile ductules in chronic viral hepatitis and subsequent postnecrotic cirrhosis was invariably observed as a regenerative reaction of the liver to injury. The extent of the ductular reaction was proportional to hepatic cell destruction. Histochemical visualization of succinic dehydrogenase was useful for the differentiation of the proliferated bile ductules originated from the trapped degenerative hepatic cells in connective tissue. Thus the term of pseudotubuli may be used with an authentic finding.
The nature and origin of the proliferated bile ductules in cholangiolotic hepatitis seemed somewhat different from that of chronic active hepatitis.

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The regenerative nodule in experimental rat cirrhosis induced by a choline deficient diet is defined as a cell mass projected on the surface of the fibrous liver. On microscopic examination, the cell masses are consisted of a small non-fatty or less fatty cells alined in one cell thick plate. They are the cells of homogenous in the property with reduced succinic dehydrogenase activity. Occasionally another type of the regeneration nodules was also encountered. The nodules are consisted of the cells of not homogeneous in the property. A large cells having a large nuclei with more than two nucleoli are arranged in rather odd fashion. Thus having a structure of adenoma. Both types of the regenerative nodules formed in fatty liver cirrhosis and proliferate expansively. Customarilly, such newly formed cell mass have been designated as the “regenerative” nodele. However, the mechanism operating the proliferation of the cells has recently debated by some investigators. A comparative study has been attempted in connection with the study of hepatic cell proliferation after partial hepatectomy in the normal liver by using a cytochemical method, tracing method using radioisotope P³², autoradiography using tritiated thymidine, however, no report came out to date for the elucidation of the subject using microspectrophotometry. The results obtained here were based on a study applied a microspectrophotometric method for the determination of DNA content of both the cells of regenerative nodules and internodular areas.

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Histochemical study was made of surgically resected adrenal glands with hyperadrenocorticism. Characteristics of lipid distribution were described in each hyperplastic adrenals in Cushing's syndrome, aldosteronism and adrenogenital syndrome. In these investigations some relationship between the histochemical change and hormone secretion in the hyperplastic cortex was also discussed.

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It has been said that the lipid content of the adrenal cortex is depleted after long standing cortisone treatment, and there is the change of lipid droplet in zona fasciculata which seemed to be changed by inhibition of ACTH production due to administration of corticoid. In our observations, marked changes were noticed on the lipid droplets of the z. fasciculata. In first 5 to 7 days, droplets became large in size, and in next 7 to 21 days, they became smaller in size and decreased their numbers, and then they disappeared. We agree to a depletion of lipid, but in our experiments we noticed that lipid droplets became larger in size before depletion. It has been said that the appearance of hydrolytic enzymes show to occur pari passu with the loss of lipids, and that the loss of lipid in various conditions associates with alterations of enzymatic distribution. In our observation, alkaline phosphatase activity localized in endothels on z. fasciculata of adrenal cortex, and this activity showed related changes with lipid droplets. If lipid droplets became large in the z. fasciculata, endothelial Alph activity disappeared, and if lipid droplets became small or disappeared, they showed more marked activity. From these findings, the authors believe that, there are some relationships between the disappearance of lipids and the endothelial Al-ph activity. It has been suggested that most important roles of G6PDH in the adrenals is to act as a generator of TPNH, which is important for hydroxylation of the steroid molecule. In our experiments, G6PDH activity in the fasciculata decreased following the administration with corticoid. TPN activity changed follwing the appearance of lipid. It has been said that adrenal of corticoid-treated rats show a decreased endogenous respiration, and that in electron microscopic studies there are the changes on the mitochondria in rat adrenocortical cells. On the whole of the enzymatic activity with respiration, we did not agree these opinions because our experiment about respiratory enzymes was not noticeable.
Our thankes are due to professor K. Akazaki and Assist. prof. N. Sasano.

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As a forestep to study the various diseases of the human adrenal gland, the adrenal glands of rat were investigated with several histochemical stainings after various treatments of the animals.

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The conducting system of the heart has been studied in detail using histological methods and wax reconstruction. In recent years histochemical observations have led to a specific tissue of knowledge concerning such aspects as glycogen concentration, enzyme activities etc. (Schiebler 1953, 1955). Though the iodine solution method (Taufic et al. 1954, Allen et al. 1959 etc.), indian ink method (Esmond et al. 1963) and fluorescine one (Iwa et al. 1961) have been reported recently, these are not always suitable to observe the peripheral ramification of the cardiac conducting system macroscopically as discussed by Otsuka and Hara (1964). The purpose of the present work was to develop a stain for the system that would be easy to use, have permanence, and be applicable to fixed material.

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In an attempt to establish the histochemical changes that occur in early myocardial infarcts, a series of experimental myocardial infarcts of predicable location was produced in 10 rats and the tissues from the normal and injured myocardium at intervals from 6 hours to one week were examined by various histochemical techniques as GOT, SDH and LAP. The earliest changes were recognized in infarcts within the first few hours as to a marked reduction of SDH and GOT of heart muscles. When a healing of necrotic myocardium by the proliferation of fibrous tissues has started, a prominent increase of LAP activity are observed within myocardial infarcts.

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In regard to morphology and function of cultured astrocytes, these were discussed from histochemical point of view. It was concluded that each type of astrocytes had different expression of metabolic patterns.

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Relating to thiamine complex effects on regeneration metabolism of nerve cells in vitro and in vivo, thiamine propyl disulfide incorporation of the spinal cord of rabbits and mice was investigated by means of autoradiographic technique.
The thiamine propyl disulfide incorporation procedure in nerve cells differs from each other between centrifugal passways, centripetal passway and cornu anterius. Though incorporating procedure should not mean utilizing procedure, it should be said that thiamine part of administrated thiamine propyl disulfide penetrates into nerve cells and plays a role in the function and metabolism directly.

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Histochemistry of primary cerebral lipidosis is not a completely explored out field of research. Many points of ambiguity and lack of information remain in literatures which cause discrepancy of opinions among authors. We recently experienced one case of late infantile form of amaurotic family idiocy (Bielschowsky), the clinical and autopsy record of which is here by preliminarily reported.

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Brain edema is believed to occur not only in the cases of cerebral diseases, but also in others such as uremia, hepatic coma and heart failure. Although it is very important problem to study its pathogenesis even in clinical medicine, it has not come to a sufficient understanding.
The studies on brain edema at the initial stage were carried out mainly from the standpoint of morphology, then extended to the physical and chemical ones, including electron microscopic study. Furthermore, histochemical method has been introduced in this field lately. In Japan also, Ambo reported some histochemical studies of brain edema concerning some enzyme activities.
On the other hand, there have been many ways to produce a experimental brain edema on animal. Since Magee's report in 1957 using Alkyl Tin compounds for this purpose, many reports of pharmacological properties on these agents were followed. Thus Aldrige and Cremar found that triethyl tin compound (TET) induced mitochondrial swelling in the liver cell and inhibited its oxidative phosphorylation.
We also have used TET in the experimental study of brain edema, esp. from the bio-and histochemical standpoint. Our findings, so far, are as follows;
1) Water, sodium and chloride contents in brain tissue incresead, while potassium showed a only slight decrease.
2) Histologically, light microscope showed few changes in superficial layer of cerebral cortex, but the perivascular and pericellular spaces were found to be dilated progressively in deeper layers, and in the white matter a prominent vacuolisation of various size was seen, resulting in sponginous appearance.
3) Electron microscopy proved no apparent changes on the capillary wall, but huge cavitation between splitted lamellae of the myelin sheath.
The present paper is to investigate some histochemical aspects of brain edema, comparing with the biochemical analysis.

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Nach der intravenösen Injektion einer schwach alkoholischen alkalischen Dithizonlösung färbt sich die Rindenplatte des Gyrus dentatus und der Felder h₃, h₄ und h₅, des Ammonshornes von Säugetieren intensiv rot (Maske 1955, Fleischhauer et al. 1957, Otsuka et al. 1962). Dieselben Gehirnanteile reagierenmit der Sulfid-Silber-Methode stark positiv (Timm 1958, Otsuka et al. 1962). Die obenzitierten Autoren meinen, daß die Dithizon- und Methode wegen der Existenz von Zink in der Ammonsformation positive Reaktionen geben. Jedoch ist es heute noch fraglich, ob die durch die beiden Methoden darstellbaren Substanzen tatsächlich dem Zink entsprechen.
In der vorliegenden Studie soil über die Lokalization von Zink im Ammonshorn mit Hilfe der Autoradiographie mit Zn⁶⁵ berichtet werden. Anßerdem wird die quantitative Verdänderung der Sulfid-Silber-Methode-positiven Substanzen unter den experimentellen Bedingungen (Dithizon-Zufuhr) untersucht.

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1. Although it is believed that ceroid and lipofuscin belong to the same system and are produced by the autoxidation of unsaturated fatty acids, and moreover, lipofuscin is far more oxidized than ceroid, it is impossible to distinguish clearly these two substances in a certain oxidation stadium. They are regarded as complex substances of chemical structures varying according to the various organs of different species of animals.
2. The lipo-pigments in the livers, in general, tend to be oxidized more rapidly than those in the brains. The lipo-pigments in the livers of aged people, for example, presented the characteristics of lipofuscin, while the lipopigments in the brains of the same subjects were observed to be of ceroid type. Chemical differences due to the locality in the brain were not ascertained. The lipo-pigments in the brains of rats with vitamin E defciency were also of ceroid type, but the livers had lipo-pigments with the intermediate characteristics between ceroid and lipofuscin. Although the lipo-pigments in the brains and livers of hogs with yellow-fat disease had similar intermediate characteristics, more marked properties of lipofuscin were observed in the livers.
3. The fat in the lipo-pigments, in accordance with the progress of autoxidation, changed from neutral to acid, while gradually losing the characteristics of fat.
4. The lipo-pigments included no free portein, but the SS and SH group reactions were positive.
5. Judging from the fact of the early appearance of lipo-pigments in rats with vitamin E deficiency, it is convincing that vitamin E deficiency accelerates the autoxidation of unsaturated fatty substances and the production of the lipo-pigments. It was not possible for histochemical differentiation pertaining to the lipo-pigments in vitamin E deficiency, yellow-fat disease, and in the senile process to be made. It is assumed that these substances in various state belong to the identical system histochemically. From these findings it can be concluded that vitamin E deficiency causes an accelerating action on the process of aging.

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