Histochemistry of calcium oxalate has not been definitive. The present author has critically investigated various histochemical methods for calcium salts and finally found a new method advantageous to those hitherto reported. Celloidin models of calcium salts, rat kidneys of oxalosis induced with ethylene glycol and human materials were used for study. New method: Firstly calcium phosphate and carbonate are removed by immersing sections into 5% acetic acid for 30 minutes. Secondly the sections still containing calcium oxalate are made to react with 5% aqueous silver nitrate for 15 minutes, and thirdly the reaction product appears dark brown to black with the use of rubeanic acid (saturated rubeanic acid in 70% alcohol with 2 drops of strong ammonium for 1 minute) that is very sensitive to silver without loosing the definite localization of calcium oxalate. This method has a merit; it does not depend on weather or illumination and takes a shorter time than the Kóssa method. A confident histochemical method for identifying calcium oxalate was established by combining this method with other characteristics of calcium oxalate such as the unstainability with hematoxylin, birefringency, solubility after incineration and gypsum formation. In the present study calcium oxalate was also found to react with lead besides silver and to appear brownish-black when one uses lead nitrate and ammonium polysulphide.
One thousand autopsy cases were studied in regard to the pathological factors of renal crystalline deposition, using the histochemical method for calcium salts by the present author, and the grade of its deposition was quantitatively determined (-∼+++). 1) Calcium oxalate crystals (CaOX) and leucine like crystals (LLC), which have both birefringency and have been often confused, were confirmed to be distinguished. This study clarifyed that the LLC presented an isogyre by a polarizing microscope and was dissolved in 5% potassium hydroxide being different from the CaOX. 2) Two hundreds cases with birefringent crystals among 1000 were divided into three groups: CaOX group 67 cases (6.7%), CaOX+LLC group 28 cases (2.8%), LLC group 105 cases (10.5%). 3) The incidence of the crystalline deposition was lower under 16 years of age and no sex difference was observed. 4) Lesions with a high incidence among 200 cases were renal and hepatic, regressive changes (metabolic disorders), hydrops of cavities and malignant neoplasms. Renal tubular degeneration was found particulary to have a high incidence and was regarded as an important factor for crystalline deposition. 5) Grade (+++) was found in the highest incidence in the CaOX group, in which the renal diseases were dominant, and grade (+) was the highest in the LLC group, which seemed to have a close relation to hepatic lesions with jaundice. The CaOX+LLC group was intermediate between both groups. 6) Cases with malignant neoplasms were found in about one half of the 200 cases, however, no difference in the incidence of crystalline deposition between cases with and without malignant neoplasms was detected.
Ultracytochemical methods for the demonstration of reactive amino groups in the cell were presented. The method utilizes either the hydroquinoneferricyanide system or the hydroquinone-tetrazolium system. The reactive amino groups were observed in the mitochondrial matrix and probably in substances in Z in the cardiac muscle of the normal adult albino mice.
In the site of steroid production, the histochemical activity of secondary alcohol dehydrogenase on isopropanol as its substrate with NAD as its coenzyme will reflect the inversion of 20α-22R-dihydroxycholesterol in the biosynthesis of steroid hormone, We applied this method on organs from all parts of the body of Wistar strain rats and observed positive reaction in the adrenal cortex, ovary and testis. In the adrenal cortex of mature rats, the zona glomerulosa exhibited a weakly positive activity, the outer to middle portions of the zona fasciculata an intensely positive activity, the inner portion of the zona fasciculata a weakly positive or negative activity, and the zona reticularis a positive activity. A proportional relationship between secondary alcohol dehydrogenase activity and the amount of lipid was present in the adrenal cortex of mature rats. In addition, the specific distribution of young and old rats was described.
DNA synthesis has been studied on the liver cells of mice in the restitution period after starvation. Tritiated thymidine (3H-TdR) was subcutaneously injected twice a day for ten days during the restitution period, in order to examine the incorporation of 3H-TdR into hepatic nuclei and the persistence of 3H-labelled nuclei in the liver. After ten-day's labelling, about 40% of hepatic parenchymal cell nuclei was heavily labelled. More incidence of heavily labelled nuclei was observed at 1 week after the last injection of 3H-TdR. Thereafter, the percentage of heavily labelled nuclei remained nearly constant around 30%. If lightly labelled nuclei were taken into account, almost 80% of the parenchymal cell nuclei showed labelling of DNA. With exception at 1 week after the last dose of 3H-TdR, this total percentage of labelled nuclei remained unchanged about 60% for 6 weeks. The incorporation of 3H-TdR into littoral cells of the liver was also observed. Throughout the present experimental period from 35 to 40% of littoral cells had labelled nuclei. These results indiacte that DNA synthesis has occurred in the parenchymal and in the littoral cell nuclei during the restitution after starvation, and that the nuclei, which contain newly syntesized DNA, persist for a long period.
When the frozen sections of glutaraldehyde- or formol-fixed tissues were incubated in lead solution and, then, converted to sulfide, marked lead staining was occurred at most of the nuclear pores in fine structural level. This characteristic seemed to be general in various cells from mammals to cold blooded animals. The organs examined were the rat liver, heart, kidney, lung and pancreas, transplanted mammary tumor of mouse, and the liver of trout. Testing with the Markham's rotation technique, the lead precipitate was mostly localized in inner and inter space of the lamellar subunits, and moderately in outside perimeter. This affinity of nuclear pore to lead ions was abolished with pretreatment of the sections in organic solvents and Triton-X-100 suggesting lipid-like nature which might regulate the interaction between nucleoplasm and cytoplasm.