Biochemically identified cancer-associated alkaline phosphatase (orthophosphoric monoester phosphohydrolase, EC 18.104.22.168) isozymes were studied by electron microscopic cytochemistry in four human cancer cell lines, as follows: chorion isozyme in KMK-2, Regan isozyme in CaR-1 and HeLa S3-10, and Kasahara isozyme in HeLa S3-5. Using ultracytochemical techniques, the chorion isozyme in KMK-2 cells was demonstrated almost exclusively on the microvilli facing the culture medium and not on the lateral surface adjacent to the neighbouring cells with interdigitating folds. In contrast, in both the HeLa sublines, which are thought to represent more immature cells, the Regan and Kasahara isozymes were demonstrated on the whole plasma membrane. The Regan isozyme in CaR-1 cells was strongly demonstrated in the intracytoplasmic microcysts in addition to the whole plasma membrane. These localizations might be related to the differentiation and deviated formation of the plasma membrane in cancer cells.
The localization of amylase and deoxyribonuclease (DNase) in rat parotid gland was investigated on both light and electron microscopic levels by use of the immunohistochemical method. Acrylamide gel embedding method was developed to prepare both ultrathin sections for electron microscopic observation and the sections for light microscopy from an identical tissue block. Both amylase and DNase localized in the secretory granules of all acinar cells at a constant concentration, but they were not recognized in duct cells. Under electron microscopic observation, these enzymes were distributed in the secretory granules and condensing vacuoles of acinar cells. Both enzymes were demonstrated simultaneously in the identical secretory granules by using immunoferritin and immunoperoxidase methods.
Sprague-Dawley male rats aged 70 days were injected with Streptozotocin with nicotinamide and then administered glucocorticoid at the ages of between 150 and 230 days. Multiple hyperplasia of the nesidoblastic cells were seen often at the age of 510 days, in addition to many islet cell tumors. As well as the induced β cell tumors, the presence of insulin antigen was detected intensively in such a nesidoblastosis by the indirect immunoperoxidase technique.
Rat thyroid gland tissues, fixed in various fixatives, were stained by means of the periodic acid thiocarbohydrazide silver proteinate (PA-TCH-SP) method of Thiéry and observed at the ultrastructural level. In electron microscopic observations, the cell organelles showing positive reactions were the Golgi apparatus, subapical granules, reabsorbed colloid droplets, lysosomes of follicular epithelial cells and colloid of the follicular lumen. The effect of fixatives was compared with respect to the differences in stainability. In each tissue fixed either with glutaraldehyde and osmium tetroxide or osmium tetroxide, only a trace of the silver reaction product was discernible. One hr treatment of TCH was not sufficient for tissues fixed singly with either paraformaldehyde or glutaraldehyde, and the stainability was strongly intensified by prolongation of TCH immersion, especially in tissues fixed only with paraformaldehyde. To intensify the PA-TCH-SP reaction, “protonation” with acetic acid and “etching” using potassium hydroxide were performed on the sections fixed singly in osmium tetroxide or glutaraldehyde, respectively. Favorable results were obtained from each treatment. Though the PA-TCH-SP method is analogous to the PAS reaction in principle, it seems that the reaction is influenced by the kind of fixative and the amount of reactive sugar.
Renal glomerular function cannot be attributed solely to structural elements in the glomerular capillary wall. Such structural elements certainly contribute to barrier function during ultrafiltration, but normal hemodynamic conditions are also required for optimal ultrafiltration. In order to change the normal hemodynamic state of glomerular capillary, an experimental congestive kidney was made by clipping unilateral renal vein in rabbits. The glomerular basement membrane was observed morphologically and cytochemically (performic acid-phosphotungstic acid reaction, PFP reaction) at various time intervals. The glomerular basement membrane in the congestive kidney showed a localized or widespread thickening and polypoid changes. Cytochemical findings were similar to that of controls.
For preservation of the cellular relationships in the specimens, freshly cut edges of mouse lymph nodes were lightly contacted onto collodion membrane covering copper grids. Air-dried spreads and those fresh frozen dried were examined by high resolution electron microscopy. Macrophages surrounded by mononuclear cells had irregular large nucleus and lucent cytoplasm containing a number of homogeneous dense bodies, mitochondria with fine dense granules and peripheral vacuoles. At higher magnification, several homogenous dense bodies were disclosed to be full of ferritin particles. These were also dispersed among ribosomes. Energy dispersive X-ray microanalysis of the homogeneous dense bodies revealed phosphorus, sulphur, chlorine and potassium. That of ferritin containing bodies indicated iron as well as these elements.
Succinic dehydrogenase activity of the 15 to 20 day murine (CD-1) mandibular joint was determined by incubating cryostat sections at 37°C for one hr in a medium containing sodium succinate. A series of adjacent sections were incubated in a medium from which the substrate had been deleted. The SDH activity in the mandibular joints of 15 and 16 day embryos was minimal and thereafter a gradual increase in the enzyme activity was evident. The cells involved in intercellular matrix elaboration (chondroblasts and osteoblasts) and those concerned in remodeling of existing structures (osteoclasts/chondroclasts) demonstrated high levels of enzyme activity. The activity observed in chondrocytes varied between slight and moderate, however, the osteocytes were non-reactive. Intense SDH activity was observed in the medial and lateral pterygoid muscles as well as in the trigeminal ganglia.
Localization of human chorionic gonadotropin (HCG) at the nidatroy sites of tubal pregnancies was studied using immunoperoxidase and immunofluorescent methods. In the villous trophoblast including anchoring villi, HCG was exclusively located in the syncytiotrophoblast. The amount of HCG which syncytiotrophoblast contained decreased in relation to the degenerative changes of the placental villi. In the anchoring villi, HCG was shown to exist more at the proximal part of the syncytiotrophoblast than at the distal part of the same villi. A markedly strong immunoreaction was observed at the syncytiotrophoblast which was scattered in the trophoblastic shell and also partly covered the trophoblastic shell. Not only the mononuclear trophoblast with abundunt glycogen in the cytoplasm, which usually existed in the trophoblastic column and shell and was classified as transitional trophoblast based on ultrastructural features, but also the mononuclear trophoblast that existed in the fibrinoid layer showed a weak but not negligible immune reaction. The characteristic localization of HCG at the nidatory site of the tubal pregnancy seemed to imply some special roles of HCG on nidation and various phenomena correlated with it. And it was also shown from these findings that the transitional trophoblast contained a small amount of HCG and that HCG concentration of the villous syncytiotrophoblast reflected the degenerative changes of the villi.
The retinas of mullet (Mugil cephalus) and carp (Cyprinus carpio) were processed for dopamine fluorescence, and were simultaneously counterstained with methyl green to obtain a differential nuclear fluorescence of other classes of cells. In tangential cryo-sections, the density was estimated for dopaminergic cells and other cells located at the innermost plane of the inner nuclear layer. In both the fish, the densities of the two classes of cells and the ratio of dopaminergic cells to other cells (mainly amacrine cells) were found to be higher in the peripheral region than in the rest of the retina.