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.

Field Emission Scanning Electron Microscopy Of Mouse Cerebellar Synaptic Contacts

2000 ◽  
Vol 6 (S2) ◽  
pp. 844-845
Author(s):  
O.J. Castejón ◽  
R. P. Apkarian ◽  
H. V. Castejón
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Field Emission ◽  
Cerebellar Cortex ◽  
Osmium Tetroxide ◽  
Phosphate Buffer Solution ◽  
Buffer Solution ◽  
Solution Ph ◽  
Osmium Tetroxide Solution ◽  
Scanning Electron

Samples of albino mice cerebellar cortex were processed by the cryofracture method for scanning electron microscopy and examined with the field emission scanning electron microscope (FESEM). Albino mouse cerebellar cortex was excised, cut into 1-2 mm slices and inmersed in 4% glutaraldehyde in O. l M phosphate buffer solution, pH 7.4, for 24h at 4°C; and postfixed for 1 h in a similarly buffered 1% osmium tetroxide solution. Specimens were dehydrated in a graded serie of ethanol (30, 50, 70, 80, 90 2x100%) prior to wrapping individual tissue pieces in preformed absolute ethanol filled parafilm cryofracture packets. Rapid freezing of packets was performed by plunging into LN2. First, the packet was transferred from the LN2 storage vessel with LNT chilled forceps in order to avoid themial damage. Secondly, the cooled fracture blade was removed from the LN2, the packet was orientated under the blade, and immediately struck with a heavy tool.

Dynorphin A(1-8): stability and implications for in vitro opioid activity

1998 ◽  
Vol 76 (3) ◽  
pp. 325-333 ◽  
Author(s):  
K M Bell ◽  
J R Traynor
Keyword(s):  
Guinea Pig ◽  
Opioid Receptors ◽  
Vas Deferens ◽  
C6 Glioma Cells ◽  
Guinea Pig Ileum ◽  
Dynorphin A ◽  
Mouse Vas Deferens ◽  
Peptidase Inhibitors ◽  
Delta Receptors

The opioid binding profile and in vitro activity of the endogenous opioid peptide dynorphin A(1-8) have been studied. At opioid receptors in guinea-pig brain dynorphin A(1-8) was nonselective, although with some preference for the delta receptor (Ki 4.6 nM) over µ (Ki 18 nM) and kappa (Ki 40 nM) receptors. However, a high degree of metabolism was observed, with less than 10% of added dynorphin A(1-8) remaining at the end of the binding assay. In the presence of peptidase inhibitors to prevent breakdown of the N- and C-termini and the Gly3-Phe4 bond the major metabolite was [Leu5]enkephalin (representing 49% recovered material). This was reduced by inclusion of an inhibitor of endopeptidase EC 3.4.24.15. In the presence of all the peptidase inhibitors the affinity for kappa receptors (Ki 0.5 nM) relative to µ and delta receptors increased, but no selectivity of binding was observed. This lack of selectivity was confirmed using membranes from C6 glioma cells expressing rat opioid receptors. The agonist effect of dynorphin A(1-8) in the mouse vas deferens (EC50 116 nM) and guinea-pig ileum (EC50 38 nM) was mediated through the kappa receptor as evidenced by the rightward shifts afforded by the kappa -selective antagonist norbinaltorphimine. In the presence of peptidase inhibition potency was improved 2-fold in the mouse vas deferens and 20-fold in the guinea-pig ileum, but this agonist activity was mediated through delta receptors in the vas deferens and µ receptors in the ileum, as a result of the formation and stabilization of [Leu5]enkephalin. The results confirm the absence of receptor selectivity of dynorphin A(1-8) in binding assays but show that its agonist effects, at least in vitro, are mediated exclusively through the kappa opioid receptor.Key words: dynorphin A(1-8), opioid receptors, peptide metabolism, mouse vas deferens, guinea-pig ileum.

scholarly journals ARTIFACTUAL LOCALIZATION OF FERRITIN IN THE CILIARY EPITHELIUM IN VITRO

1965 ◽  
Vol 25 (2) ◽  
pp. 1-7 ◽  
Author(s):  
John Mcd. Tormey
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Basement Membrane ◽  
Osmium Tetroxide ◽  
Colloidal Particles ◽  
Ciliary Epithelium ◽  
Albino Rabbit ◽  
Cytoplasmic Matrix ◽  
Tracer Particles

The accumulation of ferritin by the ciliary epithelium of the adult albino rabbit has been studied by electron microscopy. The experiments have been carried out under in vitro conditions, such that any uptake observed should be the result of passive diffusion of the tracerparticles rather than the product of active metabolic processes. The cells were fixed in osmium tetroxide and embedded in Araldite. Ferritin was found localized in three areas: in rows of apparent vesicles, free in the cytoplasmic matrix, and in the basement membrane. Some of the conclusions reached are as follows. The appearance of tracer in rows of vesicles is not in itself an adequate demonstration of pinocytosis. The permeability of the plasma membrane is drastically increased by osmium tetroxide fixation, so that tracer particles are free to diffuse across the membrane and wander through the cytoplasm. These results indicate the serious danger of being misled by artifacts when colloidal particles are used as tracers.

scholarly journals CRITIQUE ON THE K-PYROANTIMONATE METHOD FOR SEMIQUANTITATIVE ESTIMATION OF CATIONS IN CONJUNCTION WITH ELECTRON MICROSCOPY

1972 ◽  
Vol 20 (1) ◽  
pp. 65-78 ◽  
Author(s):  
RICHARD L. KLEIN ◽  
SHYUE-SHONG YEN ◽  
ÅSA THURESON-KLEIN
Keyword(s):  
Electron Microscopy ◽  
Divalent Cations ◽  
Test Tube ◽  
Tetraacetic Acid ◽  
Test Conditions ◽  
Solubility Products ◽  
Controlled Preparation ◽  
Preparation Techniques ◽  
Potassium Pyroantimonate

The histochemical method employing potassium pyroantimonate in conjunction with electron microscopy has been investigated using carefully controlled preparation techniques and very sensitive atomic absorption analysis of cations. A critique on the reliability and limitations of the method based on test tube and in vitro experiments is given. The method is sensitive to Ca++, Mg++ and Na+ at the 10–6, 10–5 and <10–2 M levels, respectively. Under defined conditions a linear ~l:l ratio of cation present to cation precipitated occurs above these levels. Approximate solubility products have been estimated. Under the test conditions, K+ does not precipitate as a pyroantimonate salt, and neither K+ nor OsO4 influences cation precipitaton at physiologic concentrations. Unbuffered, Tris-HCl-buffered and weakly buffered NaHCO3 media at pH 7.2-7.8 give statistically similar results with Na+ precipitation. The pyroantimonate ion can compete with chelators, ethylenedinitrilotetraacetic acid and ethylene glycol bis-N, N'-tetraacetic acid, for divalent cations when employed simultaneously. These chelators effectively remove Ca++ but not Mg++ from embryonic myocardium, and their effects on Na+ and K+ balance are not marked if employed for relatively short periods. Electron micrographic examples of cation precipitates are given in support of certain findings. A brief discussion of the significance of pyroantimonate grain size, the discrepancy between the ratio of intra- and extracellular precipitates and guidelines for the use of the method are included.

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.

Ultrahistochemical Studies of Mucosubstances : Electron Microscope Observation and Microprobe Analysis of the Chick Embryo Cornea

1973 ◽  
Vol 31 ◽  
pp. 344-345
Author(s):  
T. Ohkura ◽  
H. Iwatsuki ◽  
T. Watanabe
Keyword(s):  
Chick Embryo ◽  
Electron Probe ◽  
Osmium Tetroxide ◽  
Microprobe Analysis ◽  
Periodic Acid ◽  
Solution Ph ◽  
Osmium Tetroxide Solution ◽  
Glutaraldehyde Solution ◽  
C 50 ◽  
Tissue Specimens

As described in a previous report, Alcian blue provides the selective contrast enhancement of interfibrillar substances of the mesostroma of the chick embryo cornea. This finding was also verified by the electron probe microanalysis of adjacent sections. In the present study the nature of interfibrillar substances was ultrahistochemically examined by a modified Seligman's method for the demonstration of some oxidizable glycols.Strips of the 4th day chick embryo.cornea were fixed in ice-cold 2.5% glutaraldehyde solution (pH 7.4). Tissue specimens treated with 1% aqueous periodic acid or 0.2% hyaluronidase solution of pH 6.0 for 15 min were transferred into 1% thiocarbohydrazide, followed by a rinse in 1% osmium tetroxide solution (60°C, 50 min).

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.

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