Bone marrow specimens were removed from the femurs of gerbils perfused with 10% buffered formalin. They were embedded in methacrylate and sectioned on a Sorvall JB-4 microtome at 1 to 3μm at 0.5μm intervals. Staining was accomplished with methylene blue-azure B-eosin as follows: Slides were immersed in the methylene blue-azure B mixture for 1min, rinsed in 95% ethyl alcohol, immersed in ethanol eosin, decolorized in acid alcohol, and dehydrated in 100% methyl alcohol, acetone and xylene. The entire staining procedure takes about 10min and yields results comparable to those derived from Wright-Giemsa stained film preparations. It allows easier and more reliable identification of the various members of the myeloid and normoblastic cell lines than hematoxylin and eosin stained bone marrow sections.
Fluorescence spectra of a blood clot formed at the cut edge of the tail-fin of goldfish were examined, with particular attention to the fluorescence of 5-hydroxytryptamine (5-HT). An attempt was made to use HCl and o-phthalaldehyde (OPT), which are known to intensify the fluorescence of 5-HT. Prior to the study on blood clot fluorescence, the fluorescence spectra of 5-HT in solution were examined at various HCl concentrations with or without the addition of OPT. Maximal fluorescence of the blood clot appeared approx. 10 min after cutting the fin, and an emission maximum with a 470 nm peak was obtained by excitation at 360 nm in 0.1 N HCl and 0.005% OPT. This spectral property was similar to that obtained with 5-HT in solution.
Three types of commercially available test kits, used routinely in the colorimetric determination of human serum cholesterol, were applied to histochemistry. All kits tested were applicable to the homogenates of the adrenal gland, brain, adipose tissue, heart, liver and pancreas of the rat. Determinar TC (Kyowa Hakko), Cholescolor 500 (Ono Yakuhin) and Biochem. Test Combination (Boehringer Mannheim) were comparable in the determination of total cholesterol in these homogenates. The highest concentration of cholesterol was detected in the adrenal gland, followed by the cerebrum, adipose tissue, liver, heart and pancreas. When applied to unfixed slices of the brain, only with Determinar TC was a satisfactory result obtained. However, the colour complex developed with the enzyme method on these sections was unable to be recognized under a light micriscope. A modification of Determinar TC, i. e., a mixture (2: 1) of the enzyme solution without a colour developer and of diaminobenzidine (60mg/ml) solution, was applied to cryostat or tissue-sectioner sections of glutaraldehyde-fixed tissues. The adrenal cortex and brown adipose tissue stained most intensely. A drop of OsO4, when applied to a stained section, markedly accentuated density. With an elimination of ingredients to disperse the colour complex and with increased peroxidase, this method could be further modified for application to electron microscopic cytochemistry.
Endogenous peroxidase activity was localized in seromucous-secretion cells of human nasal glands with the diaminobenzidine method for light and electron microscopic histochemistry. Localization of the activity occurred in the cisternae of rough surfaced endoplasmic reticulum, including the nuclear envelope, some vesicles and saccules of the Golgi complex, and secreting granules. In the case of patients with a house dust nasal allergy, peroxidase activity in the nasal glands diminished or disappeared. However, activity recovered after reception of steroid hormone (dexamethasone 5mg/day for 7days). If nasal glands rely on the same nervous system as do salivary glands, then the diminishing of peroxidase activity may be due to the diminishing of sympathetic stimulation. Therefore, the relationship between nasal allergy and the sympathetic nerve will be considered.
Exogenous horseradish peroxidase (HRP, Type VI, Sigma) was applied as a marker for retrograde neuroanatomical tracing in the nigroneostriatal system and the sciatic nerve of adult rats. The retrograde transport and intraneuronal fate of HRP was examined with light and electron microscopy. In the nigroneostriatal system, the labeled neurons containing many brown granules in their perikarya were observed in the ipsilateral substantia nigra 3-4 hours after an injection of HRP, confirming the existence of a nigroneo-striatal pathway. Ultrastructually, these granules appear as multivesicular and lysosomal bodies in the perinuclear region. The labeled nerve cells of the spinal ganglia were also observed within 12 hours after the application of HRP in the sciatic nerve. The HRP positive brown granules had accumulated in the perinuclear region and were rapidly degraded by the lysosomal system disappearing 2 weeks after the injection. In instances of long periods of survival, characteristic lysosomal bodies which included crystals, crystalloid and laminar structures were found.
The ultrastructure of cortical granules and cortical alveoli in the eggs of Japanese palolo (a polychaete, Tylorrhynchus heterochaetus) was studied electronmicroscopically, utilizing various cytochemical stainings. The dense components of the cortical granules were stained with alcoholic phosphotungstic acid (PTA) (block staining), aqueous PTA, periodic acid (PA) -silver and PA-PTA. The filamentous substance in the cortical alveoli (stored jelly substance) was also stained with aqueous PTA and PA-PTA, but not with PTA (block staining) or PA-silver. The components of both granules are discussed cytochemically.
This paper describes a new method for the geometrical demonstration of lactate dehydrogenase (LDH) isoenzymes in tissues and organs (histochemical electrophoresis). The principle of this method can be found in the following points. (i) The tissues obtained from various organs were frozen, and cut in 8μ slices with a cryostat. The sections were placed on a piece of filterpaper and dried immediately under a fan. (ii) As a result, the enzyme molecules of the tissue were resorbed in the surface layer of the filter paper. (iii) Then, the filter paper is treated by standard electrophoresis. The data obtained with this new method were compared to those of tissue extracts of the heart, liver and kidney of dogs. This new method makes it possible to investigate the distribution of isoenzymes in tissue as a whole. Therefore, it may enable us to clarify the geometrical distribution of LDH isoenzymes in various organs.
Human chorionic gonadotropin (HCG) was proved to exist on the cell surface of a syncytiotrophoblast by the immuno-electron microscopic technique. The cell surface, including microvilli and micropinocytosis vesicles, was positive. Two types of HCG were observed; one loosely and the other tightly connected to the cell surface. The latter was closely related to the acid mucopolysaccharide of the cell surface coat, revealed by ruthenium red staining. These ultrastructural findings suggest that HCG acts as a local immunosuppressant on the syncytiotrophoblast cell surface, preventing partial maternal immunological reactions against the fetus.
Young male Wistar king rats (ca. 100-150g) were kept on a vitamin E deficient diet for 8 and 15 weeks respectively. Some of the rats from the 15 week deficiency group were subsequently fed with vitamin E supplemented diet for a further 15 weeks. Ultracytochemical changes of acid phosphatase (ACPase) activity with reference to morphological changes were observed in the hepatic parenchymal and Kupffer cells of these rats against controls. In the deficient specimens, enzymatically positive and negative lysosomes were seen in the hepatic parenchymal cells, with occasional morphologically altered Golgi apparatus with no activity. In these cells, an increased number of fat vacuoles were seen. Kupffer cells, on the other hand, showed enlarged lysosomes and enormously enlarged lipid pigment bodies or ceroids with no trace of activity. Autophagolysosomes were also seen with varying degrees of activity. In the rats of a vitamin E supplemented diet, no large lipid pigment bodies of this type were encountered. Their histochemical and morphological analysis was similar to that of the controls, other than having some fat vacuoles. This indicates the morphological and enzymatic recovery subsequent to the vitamin E supplementation. The results are discussed in relation to the lipid peroxidation.
The cellular site of metallothionein, a cadmium-and zinc-binding protein, was investigated by means of immunofluorescent antibody technique in several organs of horse, human, monkey, pig, bovine, mouse and rat. The antisera to the metallothionein was raised in rabbits. A single precipitin band was observed between antisera and antigen using both agar diffusion method and immunoelectrophoresis. The material reacting with anti-equine metallothionein antisera has been detected in the renal tissue of horse, human and monkey, but not of pig, bovine, mouse and rat. Metallothionein was mainly found in the cytoplasms of proximal convoluted tubules and Henle loop of these kidneys. No remark-kable positive fluorescence was found in other organs. The concentration of cadmium in the kidneys of these animals was measured as 30-130ppm in horse and monkey, and 0.1-0.3ppm in pig, cow, mouse and rat.
Amino groups in rabbit erythrocytic membrane were ultracytochemically demonstrated using the hydroquinone-tetranitroblue tetrazolium system. Reaction products were observed within the membrane, which was immediately external to the phosphotungstic acid-positive zone, in the form of small globules or particles with generally a diameter of 3.0-9.0 nm, aligning in one or several rows. It was found by use of a goniometer stage that the membrane area with several rows of particles corresponded in reality to the tangentially cut area of that with one row of particles. The detected intramembranous amino groups seemed to have originated from proteins, probably globular proteins, rather than from carbohydrates or lipids, since reaction products detected were found within the membrane and resisted through extraction of specimens in organic solvents as well as digestion by phospholipase C, but were not found in specimens treated with protease. These findings are of great interest in relation to intramembranous particles observed in freeze-fractured or freeze-etched membranes of various cells and also to the hypothesis of the fluid mosaic model of the cell membrane.