scholarly journals ULTRASTRUCTURAL LOCALIZATION OF ACID MUCOSUBSTANCE AND ANTIMONATE-PRECIPITABLE CATION IN HUMAN AND RABBIT PLATELETS AND MEGAKARYOCYTES

1969 ◽  
Vol 17 (12) ◽  
pp. 781-792 ◽  
Author(s):  
S. S. SPICER ◽  
W. B. GREENE ◽  
J. H. HARDIN
Keyword(s):  
Bone Marrow ◽  
Osmium Tetroxide ◽  
Plasma Membranes ◽  
Ultrastructural Localization ◽  
Human Platelets ◽  
Buffy Coat ◽  
Cytoplasmic Granules ◽  
Dense Bodies ◽  
Osmium Tetroxide Solution ◽  
Potassium Pyroantimonate

For selective ultrastructural localization of acid mucosubstance in rabbit and human platelets and megakaryocytes, bone marrow and buffy coat specimens were fixed with formalin, glutaraldehyde or osmium tetroxide, sectioned at 40 µ and stained with the Rinehart-Abul-Haj solution of dialyzed iron. In specimens from both rabbit and man, dialyzed iron staining was observed within nucleoids of the cytoplasmic granules (α-granulomeres) of platelets and megakaryocytes, on the outer surface of the plasma membranes of platelets and megakaryocytes and on the luminal surface of demarcation membranes of megakaryocytes. These results were obtained following any of the three fixation procedures, except when nucleoids failed to stain after glutaraldehyde fixation. For ultrastructural localization of pyroantimonate-precipitable cation, bone marrow and buffy coat specimens were fixed in Komnick's solution of potassium pyroantimonate and osmium tetroxide. In specimens from both species, antimonate deposits were localized within the dense bodies (5-hydroxytryptamine organelles) of platelets and within nucleoids of cytoplasmic granules of platelets and megakaryocytes. The dense bodies were well preserved in platelets fixed in a pyrophosphate-osmium tetroxide solution but were poorly, if at all, preserved by osmium tetroxide solutions containing other buffers.

scholarly journals ULTRASTRUCTURAL LOCALIZATION OF DIALYZED IRON-REACTIVE MUCOSUBSTANCE IN RABBIT HETEROPHILS, BASOPHILS, AND EOSINOPHILS

1971 ◽  
Vol 48 (2) ◽  
pp. 368-386 ◽  
Author(s):  
J. H. Hardin ◽  
S. S. Spicer
Keyword(s):  
Bone Marrow ◽  
Plasma Membrane ◽  
Golgi Complex ◽  
Osmium Tetroxide ◽  
Ultrastructural Localization ◽  
Buffy Coat ◽  
Reactive Material ◽  
Cytoplasmic Granules ◽  
Primary Granules ◽  
Acid Mucosubstances

For ultrastructural localization of acid mucosubstances in rabbit granulocytes, bone marrow and buffy coat specimens were fixed with formalin, glutaraldehyde, or osmium tetroxide, sectioned at 40 µ, and stained with the Rinehart and Abul-Haj solution of dialyzed iron (DI). Heterophils revealed DI staining on the outer surface of the plasma membrane, in the Golgi complex involved in primary granulogenesis, and in primary granules. The intragranular distribution of DI-stained material varied at different stages in the maturation of primary granules. Immature granules of heterophils fixed by any of the three methods contained a peripheral concentric band of DI-positive material; however, fully mature primary granules possessed a core of DI-reactive material in heterophils fixed with osmium tetroxide, but they contained little or no staining in heterophils fixed with formalin or glutaraldehyde. Secondary granules of rabbit heterophils failed to stain with DI. Tertiary granules, observed only in late heterophils, contained distinct DI-positive particles. Basophil granules exhibited intensely DI-stained material distributed in an orderly pattern throughout the granule. In eosinophils, DI staining was localized in the Golgi complex and in the rims of a few immature cytoplasmic granules.

scholarly journals Localization of calcium in differentiating odontoblasts and ameloblasts before and during early dentinogenesis and amelogenesis in hamster tooth germs.

1985 ◽  
Vol 33 (6) ◽  
pp. 595-603 ◽  
Author(s):  
D M Lyaruu ◽  
A L Bronckers ◽  
E H Burger ◽  
J H Wöltgens
Keyword(s):  
Osmium Tetroxide ◽  
Plasma Membranes ◽  
Pronounced Increase ◽  
Ionic Calcium ◽  
Enamel Mineralization ◽  
Dentin Mineralization ◽  
Membrane Bound ◽  
Tooth Germs ◽  
Cytoplasmic Side ◽  
Potassium Pyroantimonate

Potassium pyroantimonate-osmium tetroxide cytochemistry has been used to study the distribution of ionic calcium in hamster tooth germs during cell differentiation and during early dentinogenesis and amelogenesis. Before the onset of mineralization, pyroantimonate (PA) reaction product was found in the nucleus of differentiating preameloblasts and preodontoblasts. In the predentin, it was preferentially located along striated collagen fibrils, lying perpendicular to the basal lamina. At the onset of mineralization, a pronounced increase of PA reaction product was evident in the predentin and on the plasma membrane and in mitochondria of both preodontoblasts and preameloblasts opposite the mineralizing mantle dentin. During early enamel mineralization, PA reaction product was present in the "growing" crystal ends, while in the secretory ameloblasts, most of the PA reaction product was localized on the cytoplasmic side of the apical plasma membranes and in mitochondria. When Tomes' processes developed, PA reaction product, both cytoplasmic and membrane bound, was low or absent deep in the processes, but gradually increased toward the apical terminal web. A corresponding gradient of PA reaction product was observed on the opposing enamel crystallites. From this study we conclude that both preodontoblasts and preameloblasts seem to be involved in calcium acquisition necessary for the early stages of mantle dentin mineralization. Tomes' processes seem to regulate the entry of calcium into the enamel mineralization front.

scholarly journals Ultrastructural localization of coagulation factor V in human platelets

Blood ◽  
1986 ◽  
Vol 68 (1) ◽  
pp. 244-249 ◽  
Author(s):  
JD Wencel-Drake ◽  
B Dahlback ◽  
JG White ◽  
MH Ginsberg
Keyword(s):  
Plasma Membranes ◽  
Coagulation Factor ◽  
Ultrastructural Localization ◽  
Ultrastructural Level ◽  
Human Platelets ◽  
Immunofluorescent Staining ◽  
Resting Cells ◽  
Factor V ◽  
Platelet Factor ◽  
Coagulation Factor V

Abstract The distribution and transport in platelets of human coagulation Factor V was investigated by immunofluorescent and immunoelectron microscopy. In resting intact platelets, little surface staining was observed by immunofluorescence. In permeable resting cells, punctate staining similar to that reported for fibrinogen (Fbg), thrombospondin (TSP), fibronectin (Fn), von Willebrand factor (VWF), B-thromboglobulin (BTG), and platelet Factor 4 (PF4) was observed. Double label immunofluorescent staining for Fbg and Factor V demonstrated colocalization, suggesting their presence in the same intracellular structure. Thrombin stimulation induced the appearance of larger (approximately 0.5 mu) immunofluorescent masses of these proteins which exactly colocalized. Thus, at the light level, Factor V and Fbg are localized in the same structure in resting and thrombin-stimulated cells. On the ultrastructural level, an alpha granule localization for Fbg has previously been established. We have extended our immunofluorescent observations regarding the localization of Factor V in human platelets by use of transmission electron microscopy of antibody-stained ultrathin frozen sections. In resting cells, staining of virtually all alpha granules was observed for Factor V. In contrast, consistent staining was absent from other organelles including plasma membranes, mitochondria, and vacuolar structures which may represent the open canalicular system. These data thus establish at the ultrastructural level an alpha granule localization of human coagulation Factor V.

Isolation Of Dense Bodies From Human Blood Platelets On Metrizamide Gradient

1981 ◽  
Author(s):  
F Rendu ◽  
M Lebret ◽  
J P Caen
Keyword(s):  
Plasma Membranes ◽  
Blood Platelets ◽  
Human Platelets ◽  
Release Mechanism ◽  
Inherited Disorders ◽  
Platelet Factor ◽  
Functional Studies ◽  
Dense Bodies ◽  
Serotonin Uptake And Release

In view of the prominent role of dense bodies in platelet activation suggested by the platelet dysfunctions observed in storage pool diseases, we have developed a method for the isolation of human platelet dense bodies, using mepa- crine to follow the purification.Each step of the purification (washing procedures, lysis and subcellular separation) has been controlled in order to obtain the minimum release of these granules. Platelet lysates were centrifuged on a short two step discontinuous metrizamide gradient which allowed the attainment of a high density pellet. This pellet consisted of isolated mepacrine fluorescent granules which showed the typical appearance of dense bodies by electron microscopy. The granule pellet was relatively free from plasma membranes as estimated by the remaining (3H) -concanavalin A or 125I after labelling the whole platelets before the fractionation. The low contamination by other granule populations was estimated by the different assayed markers, β-glucuronidase, monoamine oxidase and platelet factor 4. The method is simple, reproducible and allows the highest enrichment in dense bodies obtained until now with human platelets(x 170 enrichment in calcium and x 110 enrichment in (14C) 5-HT after labelling the whole platelets as compared to the homogenate). Functional studies performed with the isolated granules showed a rapid accumulation of (14C)-5-HT, and the initial uptake was inhibited by reserpine but remained insensitive to imipramine.The technique can be applied to the study of inherited disorders where the serotonin uptake and release mechanism has to be clarified.

Selective subcellular localization of cations with variants of the potassium (pyro)antimonate technique.

1975 ◽  
Vol 23 (8) ◽  
pp. 575-598 ◽  
Author(s):  
J A Simson ◽  
S S Spicer
Keyword(s):  
Osmium Tetroxide ◽  
Acinar Cells ◽  
Distribution Patterns ◽  
High Concentration ◽  
Parotid Acinar Cells ◽  
Dense Bodies ◽  
Osmium Tetroxide Solution ◽  
High K ◽  
Rat Parotid

Fixation of rat parotid with an unbuffered osmium tetroxide solution containing nearly saturated potassium (pyro)antimonate resulted in abundant deposition of cation-antimonate precipatates in acinar cells. Altering the antimonate concentration, including buffers or chelators in the solution or changing the primary fixative resulted in an altered intensity and distribution of the precipitates formed in the tissue, apparently reflecting a degree of selectivity in ion localization. Decreasing the concentration of pyroantimonate to about half-saturation preserved predominantly the less soluble antimonate salts (e.g., Na+, Ca++) and resulted in preferential retention of deposits along the plasmalemma and in mitochondrial "dense bodies," with loss of most cytoplasmic and nuclear precipitates. A similar pattern was seen if fixation with the high concentration antimonate-osmium procedure was followed by a prolonged rinse. Adding phosphate or collidine buffers markedly decreased precipitates in the nuclei and on granular reticulum as well. Phosphate buffer or ehtyleneglycoltetraacetate inhibited in vitro precipitation of calcium and sodium and decreased or abolished plasmalemmal deposits. Glutaraldehyde fixation, either in the presence of antimonate or prior to antimonate-containing osmium tetroxide, abolished heterochromatin deposits. Mitochondrial dense bodies were of two types, one containing precipitate and the other inherently osmiophilic. The latter were also observed in pyrophosphate-osmium controls. Results from in vitro titrations of cations with the various antimonate methods and from neutron activation analyses of fixed tissues supported conclusions drawn from fine structural distribution patterns and were interpreted as follows. In rat parotid acinar cells, deposits in heterochromatin and on granular reticulum probably arose from precipitation in sites of high K+ and H+ as well as--NH3+-rich histones. Plasmalemmal antimonate deposits demonstrated sites of sodium and/or calcium accumulation. Some mitochondrial dense bodies contained Ca++ whereas others were inherently osmiophilic. Large, extracellular deposits were probably predominantly sodium precipitates.

scholarly journals ELECTRON PROBE ANALYSIS OF CATIONIC SPECIES IN PYROANTIMONATE PRECIPITATES IN EPON-EMBEDDED TISSUE

1969 ◽  
Vol 17 (2) ◽  
pp. 102-106 ◽  
Author(s):  
BERNARD P. LANE ◽  
EUGENE MARTIN
Keyword(s):  
Electron Microscopy ◽  
Electron Probe ◽  
Osmium Tetroxide ◽  
Vas Deferens ◽  
Apical Plasma Membrane ◽  
Mouse Vas Deferens ◽  
Electron Probe Analysis ◽  
Osmium Tetroxide Solution ◽  
Potassium Pyroantimonate

Electron microscopy of Epon-embedded mouse vas deferens eipthelium treated with buffered potassium pyroantimonate-osmium tetroxide solution revealed precipitates in the lamina propria and along the apical plasma membrane. Electron microprobe elemental analysis of adjacent sections demonstrated that the deposits contained sodium and antimony. Other cations noted to precipitate pyroantimonate in vitro were not present in large amounts compared to controls, and were randomly distributed.

ULTRASTRUCTURAL LOCALIZATION OF A CHARACTERISTIC ACID PHOSPHATASE IN GRANULES OF RABBIT BASOPHILS

1974 ◽  
Vol 22 (12) ◽  
pp. 1092-1104 ◽  
Author(s):  
ATSUSHI KOMIYAMA ◽  
SAMUEL S. SPICER
Keyword(s):  
Acid Phosphatase ◽  
Phosphatase Activity ◽  
Acid Phosphatase Activity ◽  
Potential Role ◽  
Strong Acid ◽  
Ultrastructural Localization ◽  
Buffy Coat ◽  
Cytoplasmic Granules ◽  
Nitrophenyl Phosphate ◽  
Tris Maleate

Bone marrow basophils incubated in Gomori medium at pH 6.0-6.8 exhibited strong acid phosphatase activity suggestive of a potential role in endocytosis in one-third of the cytoplasmic granules and also in Golgi elements. Buffy coat basophils contained about one-third as many reactive granules. Reaction product was confined to the threadlike component of the larger granules predominant in early basophils and was absent from the denser-type granules predominant in late basophils. In centrioles of basophils acid phosphatase appeared localized between triplet fibers. Reactivity with the Gomori medium was diminished at pH 5.0, absent at pH 8.0 and only slightly decreased with p-nitrophenyl phosphate as substrate. Basophils incubated in Barka-Anderson medium at pH 5.0-6.8 revealed light acid phosphatase activity in the Golgi lamellae but essentially none in cytoplasmic granules. Tris-maleate buffer of the Barka-Anderson medium replacing the sodium acetate of the Gomori medium inhibited the reactivity in the granules. Incubation in media containing NaF, or lacking substrate, eliminated the heavy precipitates in granules and Golgi elements but yielded light, nonenzymatic lead staining in Golgi and tubulovesicular structures and atypical granules present only in buffy coat basophils.

scholarly journals Ultrastructural localization of coagulation factor V in human platelets

Blood ◽  
1986 ◽  
Vol 68 (1) ◽  
pp. 244-249 ◽  
Author(s):  
JD Wencel-Drake ◽  
B Dahlback ◽  
JG White ◽  
MH Ginsberg
Keyword(s):  
Plasma Membranes ◽  
Coagulation Factor ◽  
Ultrastructural Localization ◽  
Ultrastructural Level ◽  
Human Platelets ◽  
Immunofluorescent Staining ◽  
Resting Cells ◽  
Factor V ◽  
Platelet Factor ◽  
Coagulation Factor V

The distribution and transport in platelets of human coagulation Factor V was investigated by immunofluorescent and immunoelectron microscopy. In resting intact platelets, little surface staining was observed by immunofluorescence. In permeable resting cells, punctate staining similar to that reported for fibrinogen (Fbg), thrombospondin (TSP), fibronectin (Fn), von Willebrand factor (VWF), B-thromboglobulin (BTG), and platelet Factor 4 (PF4) was observed. Double label immunofluorescent staining for Fbg and Factor V demonstrated colocalization, suggesting their presence in the same intracellular structure. Thrombin stimulation induced the appearance of larger (approximately 0.5 mu) immunofluorescent masses of these proteins which exactly colocalized. Thus, at the light level, Factor V and Fbg are localized in the same structure in resting and thrombin-stimulated cells. On the ultrastructural level, an alpha granule localization for Fbg has previously been established. We have extended our immunofluorescent observations regarding the localization of Factor V in human platelets by use of transmission electron microscopy of antibody-stained ultrathin frozen sections. In resting cells, staining of virtually all alpha granules was observed for Factor V. In contrast, consistent staining was absent from other organelles including plasma membranes, mitochondria, and vacuolar structures which may represent the open canalicular system. These data thus establish at the ultrastructural level an alpha granule localization of human coagulation Factor V.

Ultrastructural localization of cationic proteins in human polymorphonuclear leukocytes

1978 ◽  
Vol 30 (1) ◽  
pp. 21-35
Author(s):  
W.J. Brown ◽  
E.M. Wood
Keyword(s):  
Polymorphonuclear Leukocytes ◽  
Osmium Tetroxide ◽  
Ultrastructural Localization ◽  
Ultrastructural Level ◽  
Cationic Proteins ◽  
Cytoplasmic Granules ◽  
Mediators Of Inflammation ◽  
Human Polymorphonuclear Leukocytes ◽  
Ammoniacal Silver ◽  
Neutral Proteases

The present investigation is concerned with the use of the post-formalin ammoniacal silver reaction to detect the arginine-rich cationic proteins in human polymorphonuclear leukocytes at the ultrastructural level. These proteins appear to function as neutral proteases in antibacterial action and as mediators of inflammation. Originally, the ammoniacal silver reaction relied upon primary fixation in dilute formalin which prevented optimum fixation of tissues. This study shows that by using the proper sequence of glutaraldehyde fixation and the ammoniacal silver solution in conjunction with osmium tetroxide treatment, better fixation of the tissue and localization of the ammoniacal silver reaction can be achieved. Also, the ammoniacal silver reaction in human polymorphonuclear leukocytes is exclusively located in the large, crystalline cytoplasmic granules of eosiniphils. All other cytoplasmic granules of neutrophils, eosinophils, and basophils were found to be devoid of the ammoniacal silver reaction product. These results are contrary to previously published experimental data. Possible explanations for this discrepancy are discussed.

Ultrastructure of Liver in Lactosyl Ceramidosis

1971 ◽  
Vol 29 ◽  
pp. 312-313
Author(s):  
Z. Hruban ◽  
J. R. Esterly ◽  
G. Dawson ◽  
A. O. Stein
Keyword(s):  
Connective Tissue ◽  
Liver Biopsy ◽  
Osmium Tetroxide ◽  
Close Contact ◽  
Multivesicular Bodies ◽  
Bile Canaliculi ◽  
Dense Bodies ◽  
Marginal Plates ◽  
Membranous Material ◽  
Perichromatin Granules

Samples of a surgical liver biopsy from a patient with lactosyl ceramidosis were fixed in paraformaldehyde and postfixed in osmium tetroxide. Hepatocytes (Figs. 1, 2) contained 0.4 to 2.1 μ inclusions (LCI) limited by a single membrane containing lucid matrix and short segments of curved, lamellated and circular membranous material (Fig. 3). Numerous LCI in large connective tissue cells were up to 11 μ in diameter (Fig. 2). Heterogeneous dense bodies (“lysosomes”) were few and irregularly distributed. Rough cisternae were dilated and contained smooth vesicles and surface invaginations. Close contact with mitochondria was rare. Stacks were small and rare. Vesicular rough reticulum and glycogen rosettes were abundant. Smooth vesicular reticulum was moderately abundant. Mitochondria were round with few cristae and rare matrical granules. Golgi complex was seen rarely (Fig. 1). Microbodies with marginal plates were usual. Multivesicular bodies were very rare. Neutral lipid was rare. Nucleoli were small and perichromatin granules were large. Small bile canaliculi had few microvilli (Fig. 1).

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