Wave dispersive X-ray microanalysis was performed on platelets and red blood cells of a healthy male and a von Willebrand patient, and mast cells and lymphocytes of the mouse peritoneal fluid. A STEM equipment of target method was modified by replacing grid holders with carbon rod tubes and the reflecting stage with a carbon disc for microanalysis, attempting to decrease the background noises. In secondary electron images at acceleration voltage of 15-45kV and absorption current of 0.03-0.1nA, the above cells were recognized and also identified in back scattered electron images at an absorption current of 0.1-0.5μA by their profiles and relationship with other cells. High peaks of characteristic X-ray counts of Na-, P-, S-, Cl and K-Kα were disclosed in the analysis of cells. Ca- and Mg-Kα were distinctly disclosed in that of the healthy male and Ca- or Mg-Kα was not in that of patient platelets. Analysis of healthy male platelets, mouse mast cells and lymphocytes by means of the pulse height analyzers detected Zn-Kα and Zn-Lα. Electron microscopy of healthy male platelets disclosed the dense bodies but platelets of the patient failed to reveal them.
To examine the intracellular localization of the pancreatic lipase in the mouse pancreas, both histochemical and immunohistochemical stainings were applied on the ultra-thin frozen sections. Furthermore, in order to study the mutual relationship of the distribution between lipase and α-amylase as well as α-amylase and chymotrypsinogen A within a zymogen granule, immunohistochemical double staining was performed. The positive reaction for lipase by the histochemical staining were observed in the peripheral zone of the zymogen granules, as belt-like electron opaque deposits of 20Å to 40Å in width. Especially, in the isolated zymogen granules, the positive reaction sites were clearly recognized inside the limiting membrane of the zymogen granule as electron dense deposits of 30Å to 40Å in diameter. By the immunohistochemical study, lipase was localized along the limiting membrane of the zymogen granule, while, α-amylase and chymotrypsinogen A were distributed diffusely in a zymogen granule. Moreover, when the isolated zymogen granules were treated with alkaline solution, large amounts of lipase remained in the membrane fraction, but most α-amylase activity (more than 95%) was extracted into the soluble fraction. This fact suggests that lipase is more closely associated with the limiting membrane of the zymogen granule, than α-amylase. From these results, the localizing pattern of the pancreatic lipase was found to be different from those of α-amylase, chymotrypsinogen A and trypsinogen; the former bound loosely to the inside of the limiting membrane of the zymogen granule, while the latter enzymes were localized diffusely within a zymogen granule.
During spermatogenesis, a great number of microtubules temporarily appear around the nucleus of the spermatid at the time the nucleus undergoes transfiguration. Therefore, the transfiguration of the nucleus is considered to be closely related to the distribution of the microtubules. In this paper, the distribution pattern of microtubules in the spermatid of the rodent, which has an asymmetrical fishhook-shaped nucleus, was compared with that of microtubules in the spermatid of the dog, which has the symmetrical ovoid nucleus. The results indicate that the microtubules in rodents were localized only on the concave side of the nucleus, while those in the dog spermatid were distributed evenly on the posterior half of the nucleus.
Polyglucose particles artificially synthesized under histochemical conditions from glucose 1-phosphate by phosphorylase and branching glycosyl-transferase in various cartilage cells were observed by light and electron microscopy. In chick embryos, the polyglucoses were stained red-violet or violet-blue with iodine, and the polyglucose particles were observed electron microscopically to be spherical branching bodies with an average diameter of 369±61.62Å. In the adult chicken, they were stained reddish-violet with iodine and formed similar particles with an average diameter of 357±65.96Å. In the human fetus, the polyglucoses were stained red-violet with iodine and the particles were observed as larger bodies with an average diameter of 529±94.98Å. In the human adult, they were stained red-brown with iodine and electron microscopic findings revealed particles which were relatively uniform and had an average diameter of 373±56.05Å. Polyglucoses synthesized in tumorous cartilage cells of human chondrosarcoma were stained red-violet with iodine and the particles were larger and more irregular in size. The average diameter was 540±169.76Å. These particles also appeared in the intranuclear regions, suggesting enzyme deviation as a result of carcinogenesis.
This study was attempted in order to devise a new embedding method for immunohistochemical studies to meet the following demands: 1) Minimum loss of antigenicities during the embedding process. 2) Simple preparation of ultrathin sections requiring no special devices. 3) Removal of embedding materials from sections under mild conditions. Acrylamide gel, cross-linked with acrylylcystamine or N, N′-diallyltartardiamide, proved to be a useful embedding media for immunocytochemistry in both light- and electron microscopy.
The presence of a myosin-like protein in rabbit oocyte is described. Fluorescent antimyosin-like protein antibodies are bound in the cytoplasm of oocyte and of granulosa cells. The findings are discussed.
The regional and cellular distribution of insulin-like immunoreactivity throughout the CNS of three rodent species were investigated using an immunofluorescence technique. No principal differences in the content and distribution of insulin-like material were revealed between lean animals and mice suffering from a genetically determined diabetes mellitus.
Observations of ACTH and TSH-producing cells of the rat pituitary studied immunohistochemically in thick sections were correlated with observations of adjacent thin sections studied by electron microscopy. ACTH-containing cells are small and stellate, with poorly developed organelles and granules ≤250nm in diameter. TSH cells are much larger, contain well-developed intracellular organelles, and variable sized granules (≤270nm), some of which appear in vesicles with dense cores. In addition, TSH cells show ultrastructural changes in response to adrenalectomy.
The ultrastructural localization of mucopolysaccharide in the pulmonary alveolus of a rat was studied by means of the colloidal iron reaction, the ruthenium red staining and the phosphotungstic acid (PTA) staining. Colloidal iron-positive material was observed in a thin layer, 15-25nm in thickness, closely attached to the luminal surface of the alveolar epithelium. The superficial layer, the hypophase, and the tubular-myelin figures, however, lacked the colloidal iron reactivity. Ruthenium red-positive material was observed on the luminal surface of the alveolar epithelium as well as in the hypophase. In the ultrathin sections stained with PTA (pH 0.4 and 1.5), the microvillous surface of the type II cell was densely stained, whereas the luminal surface of the type I cell was not discernible. The results suggest that acid mucopolysaccharide is not involved in the surfactant lining, but may be in existence as a surface coat on the alveolar epithelium; the surface coat on the type II cell differs chemically from that on the type I cell.
The ultrathin sectioning, freeze-etching, negative staining, ruthenium red staining, ultracytochemical demonstration of sulfhydryl (SH) groups and performic acid-phosphotungustic acid (PFP) reaction were performed on intact and isolated lumenal plasma membranes of the normal rabbit urinary bladder. The lumenal membrane was characterized by a scalloped configuration with the concave plaque and crest-like interplaque regions. The concave plaque regions showed an asymmetric unit membrane of 12nm in width, having a thicker outer leaflet of 6nm which is composed of periodic dots. The plaque regions consisted of the hexagonal particle array (HPA) in the EF face and the interplaque regions smooth in the freeze-replica images. HPA, 13nm in diameter, was shown to be composed of 12 subparticles of 2.5nm in diameter in the negatively stained isolated membranes. The surface coat, 5-10nm in width, was demonstrated on the lumenal surface by a ruthenium red staining. The PFP reaction revealed the trilaminar structure in the interplaque regions, whereas the density (2nm in width) spanning membrane, resembling a ladder-like structure, was observed in the plaque regions. Hexagonally arranged electron lucent areas were also recognized in tangential sections. The substances stained with the PFP reaction may represent phospholipids and glycolipids in the lumenal plasma membrane. Reaction products for SH groups in the form of particles of 3-9nm in diameter were observed in and around the membrane in both the plaque and the interplaque regions and were often deposited directly on the lumenal membrane. It is suggested that the SH groups demonstrated here are derived from the surface proteins, perhaps enzyme proteins in the lumenal plasma membrane.
Ouabain-sensitive, K-dependent p-nitrophenylphosphatase activity was studied cytochemically in photoreceptor cells of the normal adult guinea pig retina with a newly developed one step lead citrate method (Mayahara et al., 1980). In guinea pig photoreceptor cells fixed for 5min in a mixture of 2% paraformaldehyde and 0.25% glutalaldehyde, electron dense reaction products were observed on plasma membranes of the inner segments and more proximal portion, but not on membranes of the outer segments. The most intensive reaction products were evident in the pre-synaptic membranes in the outer plexiform layer. It is also noteworthy that this activity was positive on the apical villi of the Müller cells, which were spread out distal to the outer limiting membrane into interstices between the inner segments. This reaction was almost completely abolished by 10mM ouabain or elimination of K. This activity was also substrate-dependent and completely inhibited by PCMBS or preheating. In contrast to K-dependent p-nitrophenylphosphatase activity, Mg-ATPase and non-specific alkaline phosphatase activity were not observed on every part of the plasma membranes of the photoreceptor cells.