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 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 alkaline phosphatase in rat eosinophil leucocytes.

1978 ◽  
Vol 26 (10) ◽  
pp. 862-864 ◽  
Author(s):  
D M Williams ◽  
J E Linder ◽  
M W Hill ◽  
R Gillett
Keyword(s):  
Alkaline Phosphatase ◽  
Plasma Membrane ◽  
Electron Microscope ◽  
Outer Surface ◽  
Human Neutrophil ◽  
Osmium Tetroxide ◽  
Diazonium Salt ◽  
Ultrastructural Localization ◽  
Microscope Examination ◽  
Membrane Location

The ultrastructural localization of alkaline phosphatase in eosinophil leucocytes, obtained from experimentally-induced peritoneal exudates in rats, has been studied using an osmiophilic technique with 2-naphthylthiolphosphoryl dichloride as substrate, fast Blue BBN as diazonium salt and postosmication with 1% aqueous osmium tetroxide. With this method identical incubation procedures could be used for both light and electron microscope examination. Eosinophils were the only cells which contained alkaline phosphatase. The enzyme was predominantly associated with the outer surface of the plasma membrane, being present in much lower concentrations in cytoplasmic cisternae. Eosinophil granules only rarely showed reaction product. The plasma membrane location of alkaline phosphatase in eosinophil leucocytes is identical to that recently demonstrated in the human neutrophil.

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 HISTOCHEMICAL DEMONSTRATION OF SULFATED MUCOSUBSTANCES AND CATIONIC PROTEINS IN HUMAN GRANULOCYTES AND PLATELETS

1969 ◽  
Vol 17 (10) ◽  
pp. 668-674 ◽  
Author(s):  
W. B. DUNN ◽  
S. S. SPICER
Keyword(s):  
Developmental Stages ◽  
Histochemical Demonstration ◽  
Histochemical Staining ◽  
Buffy Coat ◽  
Human Neutrophils ◽  
Cationic Proteins ◽  
Cytoplasmic Granules ◽  
Late Developmental Stage ◽  
Staining Methods ◽  
Primary Granules

Histochemical staining methods visualize sulfated mucosubstance in numerous small granules of early neutrophil leukocytes in appropriately processed human bone marrow smears. These reactive granules presumably constitute the counterpart in human neutrophils of the mucosaccharide-rich primary granules in rabbit heterophils. Neutrophils at a late developmental stage in human marrow or buffy coat smears reveal few or no granules with such reactivity. Strong staining for sulfated mucosubstance is readily demonstrable in numerous large cytoplasmic granules in human eosinophils. This reactivity diminishes but does not disappear during maturation of the eosinophil and its single population of cytoplasmic granules. Granules of human basophils at all developmental stages stain for sulfated mucosubstance. Platelets in human buffy coat smears reveal evidence for sulfated mucosaccharide. The mucosaccharide-containing granules of the three myeloid series also disclose acidophilia at high pH indicative of the presence of strongly cationic proteins.

scholarly journals CYTOPLASMIC GRANULE FORMATION IN MYELOCYTES

1966 ◽  
Vol 29 (2) ◽  
pp. 307-316 ◽  
Author(s):  
Martha E. Fedorko ◽  
James G. Hirsch
Keyword(s):  
Amino Acids ◽  
Bone Marrow ◽  
Electron Microscope ◽  
Golgi Complex ◽  
Time Sequence ◽  
Granule Formation ◽  
Initial Exposure ◽  
Cytoplasmic Granules

The intracellular flow of tritiated lysine as revealed by electron microscope radioautography was studied in heterophilic myelocytes of rabbit marrow. Label over the Golgi complex rose to a maximum of 37% of total cytoplasmic grains 30 min after initial exposure to the tracer and fell to 11% after 3 to 4 hr of incubation. Coincident with decrease in label over the Golgi complex, grain counts over granules rose to 32% after 3 to 4 hr. The time sequence of incorporation and flow of tritiated lysine and the per cent distribution of label was similar in bone marrow myelocytes under in vivo and in vitro conditions. The results demonstrate a function of the Golgi complex in incorporating or packaging certain basic amino acids or proteins into cytoplasmic granules of heterophilic myelocytes.

scholarly journals Formation of Cytoplasmic Granules in Human Eosinophilic Myelocytes: An Electron Microscope Autoradiographic Study

Blood ◽  
1968 ◽  
Vol 31 (2) ◽  
pp. 188-194 ◽  
Author(s):  
MARTHA E. FEDORKO
Keyword(s):  
Bone Marrow ◽  
Electron Microscope ◽  
Golgi Complex ◽  
Autoradiographic Study ◽  
Electron Microscope Autoradiography ◽  
Cytoplasmic Granules ◽  
Microscope Autoradiography ◽  
Intracellular Amino Acids ◽  
Flow Through

Abstract The intracellular flow of tritiated lysine in human eosinophilic myelocytes was studied by electron microscope autoradiography so that information could be obtained on the formation of eosinophil granules. Bone marrow particles obtained from a patient with a marked increase in the number of bone marrow eosinophils were incubated in vitro for periods up to 150 minutes. The percentage of cytoplasmic grains over the Golgi complex rose from 11 percent at 5 minutes to 28 percent by 30 minutes and fell to 15 percent at 150 minutes. Grains over cytoplasmic granules steadily rose to 37 percent by 150 minutes. These results are statistically significant and demonstrate that: human eosinophilic myelocytes are able to form cytoplasmic granules under the in vitro conditions employed, and that intracellular amino acids or proteins flow through the Golgi complex before incorporation into granules.

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.

Ultrastructural Features of the Oral Apparatus of Stentor

1976 ◽  
Vol 34 ◽  
pp. 142-143
Author(s):  
Elizabeth F. Howell
Keyword(s):  
Plasma Membrane ◽  
Osmium Tetroxide ◽  
Buccal Cavity ◽  
Fiber Bundles ◽  
Microtubular Root ◽  
Stentor Coeruleus ◽  
Root Fiber

The ultrastructure of the normal oral apparatus of Stentor has not been extensively studied. I report here on the ultrastructure of the buccal cavity of Stentor coeruleus.Stentor coeruleus was fixed in either a buffered mixture of osmium tetroxide and glutaraldehyde, or in buffered glutaraldehyde alone. Cells were then dehydrated and embedded in a mixture of Epon and Araldite.An extensive adoral zone of membranelles surrounds the anterior of the cell, and each membranelle consists of 2 parallel rows of cilia. These extend down into the buccal cavity. Two microtubular root fibers, or nemadesmata (Figs. 2 and 5), extend deeply into the cytoplasm from the base of each ciliary kinetosome. Mitochondria are usually closely associated with the root fiber bundles, and small vesicles are present between the nemadesmata of adjacent kinetosomes (Fig. 5). In the cytopharyngeal, non-ciliated areas of the buccal cavity, microtubular ribbons which extend into the cytoplasm are aligned perpendicular to the plasma membrane of the buccal cavity (Figs. 1 and 2).

Immunogold localization of HMB-45 antigen in pulmonary lymphangiomyomatosis

1995 ◽  
Vol 53 ◽  
pp. 998-999
Author(s):  
J.M. Minda ◽  
E. Dessy ◽  
G. G. Pietra
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Room Temperature ◽  
Osmium Tetroxide ◽  
Muscle Cells ◽  
Ultrastructural Localization ◽  
Reproductive Age ◽  
Thin Sections ◽  
Smooth Muscle Proliferation ◽  
Hmb 45

Pulmonary lymphangiomyomatosis (PLAM) is a rare disease occurring exclusively in women of reproductive age. It involves the lungs, lymph nodes and lymphatic ducts. In the lungs, it is characterized by the proliferation of smooth muscle cells around lymphatics in the bronchovascular bundles, lobular septa and pleura The nature of smooth muscle proliferation in PLAM is still unclear. Recently, reactivity of the smooth muscle cells for HMB-45, a melanoma-related antigen has been reported by immunohistochemistry. The purpose of this study was the ultrastructural localization of HMB-45 immunoreactivity in these cells using gold-labeled antibodies.Lung tissue from three cases of PLAM, referred to our Institution for lung transplantation, was embedded in either Poly/Bed 812 post-fixed in 1% osmium tetroxide, or in LR White, without osmication. For the immunogold technique, thin sections were placed on Nickel grids and incubated with affinity purified, monoclonal anti-melanoma antibody HMB-45 (1:1) (Enzo Diag. Co) overnight at 4°C. After extensive washing with PBS, grids were treated with Goat-anti-mouse-IgG-Gold (5nm) (1:10) (Amersham Life Sci) for 1 hour, at room temperature.

Ultrastructural and immunocytochemical studies of the rat hypothalamo-neurohypophysial system processed by rapid freezing and freeze-substitution fixation

1990 ◽  
Vol 48 (3) ◽  
pp. 884-885
Author(s):  
Seiji Shioda ◽  
Yasumitsu Nakai ◽  
Atsushi Ichikawa ◽  
Hidehiko Ochiai ◽  
Nobuko Naito
Keyword(s):  
Osmium Tetroxide ◽  
Freeze Substitution ◽  
Ultrastructural Localization ◽  
Wistar Rat ◽  
Neurosecretory Cells ◽  
Rapid Freezing ◽  
Sodium Metaperiodate ◽  
Tissue Sections ◽  
Ultrathin Sections ◽  
Electron Microscopes

The ultrastructure of neurosecretory cells and glia cells in the supraoptic nucleus (SON) of the hypothalamus and the neurohypophysis (PN) was studied after rapid freezing followed by substituion fixation. Also, the ultrastructural localization of vasopressin (VP) or its carrier protein neurophys in II (NPII) in the SON and PN was demonstrated by using a post-embedding immunoco1loidal gold staining method on the tissue sections processed by rapid freezing and freeze-substitution fixation.Adult male Wistar rat hypothalamus and pituitary gland were quenched by smashing against a copper block surface precooled with liquid helium and freeze-substituted in 3% osmium tetroxide-acetone solutions kept at -80°C for 36-48h. After substituion fixation, the tissue blocks were warmed up to room temperature, washed in acetone and then embedded in an Epon-Araldite mixture. Ultrathin sections mounted on 200 mesh nickel grids were immersed in saturated sodium metaperiodate and then incubated in each of the following solutions: 1 % egg albumin in phosphate buffer, VP or NPII (1/1000-1/5000) antiserum 24h at 4°C, 3) colloidal gold solution (1/20) 1h at 20°C. The sections were washed with distilled waterand dried, then stained with uranylacetate and lead citrate and examined with Hitachi HU-12A and H-800 electron microscopes.

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