scholarly journals Preservation of arachidonoyl phospholipids during tissue processing for electron microscopic autoradiography.

1985 ◽  
Vol 33 (8) ◽  
pp. 799-802 ◽  
Author(s):  
C M Krueger ◽  
E J Neufeld ◽  
J E Saffitz
Keyword(s):  
Tannic Acid ◽  
Osmium Tetroxide ◽  
Specific Class ◽  
Electron Microscopic ◽  
En Bloc ◽  
Electron Microscopic Autoradiography ◽  
Tissue Processing ◽  
Cross Links ◽  
Murine Fibrosarcoma

To facilitate autoradiographic subcellular localization of arachidonoyl phospholipids, the retention of radioactivity during tissue processing of murine fibrosarcoma cells labeled in vitro with 3H-arachidonate was assessed. Approximately 94% of cell radioactivity was incorporated into phospholipids. During tissue processing, extraction of radioactivity was monitored by liquid scintillation spectrometry. Fixation of cells in glutaraldehyde-tannic acid, postfixation in osmium tetroxide, en bloc staining in uranyl magnesium acetate, dehydration in ethanol, and embedding in Epon resulted in preservation of 93.5% of total tissue radioactivity. Analysis of extracted radioactivity by thin layer chromatography revealed that no specific class of phospholipids was selectively extracted. Fixation with osmium tetroxide alone was nearly as effective as the complete fixation protocol and resulted in retention of 90.0% of radioactivity. However, fixation with glutaraldehyde-tannic acid alone without osmium tetroxide post-fixation led to extraction of 69.8% of total cell radioactivity. Thus, osmium tetroxide is crucial in the preservation of arachidonoyl phospholipids and presumably forms extensive cross-links between polyunsaturated acyl residues. This degree of preservation of arachidonoyl phospholipids is indicative of spatial fixation of the radiolabeled moieties and will permit quantitative studies of subcellular loci of eicosanoid metabolism by electron microscopic autoradiography.

Electron microscopic study of the membrane glycoproteins in the rat kidney after cisplatin treatment

1989 ◽  
Vol 47 ◽  
pp. 912-913
Author(s):  
S.K. Aggarwal
Keyword(s):  
Alkaline Phosphatase ◽  
Rat Kidney ◽  
Light And Electron Microscopy ◽  
Kidney Tissue ◽  
Membrane Glycoproteins ◽  
Electron Microscopic ◽  
Before And After ◽  
Interstrand Cross Links ◽  
Cross Links

The proposed primary mechanism of action of the anticancer drug cisplatin (Cis-DDP) is through its interaction with DNA, mostly through DNA intrastrand cross-links or DNA interstrand cross-links. DNA repair mechanisms can circumvent this arrest thus permitting replication and transcription to proceed. Various membrane transport enzymes have also been demonstrated to be effected by cisplatin. Glycoprotein alkaline phosphatase was looked at in the proximal tubule cells before and after cisplatin both in vivo and in vitro for its inactivation or its removal from the membrane using light and electron microscopy.Outbred male Swiss Webster (Crl: (WI) BR) rats weighing 150-250g were given ip injections of cisplatin (7mg/kg). Animals were killed on day 3 and day 5. Thick slices (20-50.um) of kidney tissue from treated and untreated animals were fixed in 1% buffered glutaraldehyde and 1% formaldehyde (0.05 M cacodylate buffer, pH 7.3) for 30 min at 4°C. Alkaline phosphatase activity and carbohydrates were demonstrated according to methods described earlier.

scholarly journals Comparison of surface iodination methods by electron microscopic autoradiography applied in vitro to different life-stages of Dipetalonema vitae (Filarioidea)

Parasitology ◽  
1982 ◽  
Vol 85 (3) ◽  
pp. 559-565 ◽  
Author(s):  
W. Baschong ◽  
W. Rudin
Keyword(s):  
Life Stages ◽  
Electron Microscopic ◽  
Electron Microscopic Autoradiography ◽  
Physiological Conditions ◽  
Chloramine T ◽  
Electron Microscopical ◽  
Dipetalonema Viteae

Different stages of Dipetalonema viteae (males, females, microfilariae, and 3rd-stage larvae) have been iodinated in vitro under physiological conditions by chloroglycoluril, lactoperoxidase or chloramine T. The concentrations of the catalysts were correlated with the viability of the worms. Localization of the label with the different iodination methods had been visualized by electron microscopical autoradiography. Chloroglycoluril-mediated iodination is predominantly localized on the filarial cuticle. Lactoperoxidase-catalysed iodination is less specific and chloramine T catalyses iodination in a gradient decreasing from the cuticle to inner structures. It is necessary to visualize the labelling by electron microscopical autoradiography prior to biochemical and immunological experiments to avoid the extraction of structures iodinated by leakage of the catalyst into sub-cuticular regions.

Autoradiographic Location of Protein Synthesized by Liver Slices

1967 ◽  
Vol 25 ◽  
pp. 140-141
Author(s):  
Charles A. Ashley ◽  
Theodore Peters
Keyword(s):  
Electron Micrographs ◽  
Osmium Tetroxide ◽  
Liver Slice ◽  
Adjacent Area ◽  
Electron Microscopic ◽  
Minimum Area ◽  
Secretory Pathways ◽  
Electron Microscopic Autoradiography ◽  
Direct Counting ◽  
Fasted Rats

The location, migration, and secretory pathways followed by proteins, newly synthesized by the liver, were determined by electron microscopic autoradiography. Small (3x4x0.5 mm) slices of liver from fasted rats were incubated for 2 minutes in medium (0.1 ml per slice)containing H3-4,5-L-leucine and then transferred to medium with unlabeled leucine for further incubation. Total incubation times were 2,4,10,25,40 and 75 minutes. Control slices were incubated in medium containing labeled leucine and puromycin. Slices were fixed in 4% formaldehyde (prepared from paraformaldehyde), post-fixed in osmium tetroxide and embedded on end in Epon. Autoradiographs were prepared using Ilford L-4 and Kodak NTE emulsions. Exposure times varied from 3 to 30 weeks. Parallel experiments were performed in which the total counts per minute per milligram of liver slice at each time were determined by standard direct counting techniques. Electron micrographs (magnification approximately 5,000 X) covering a minimum area of 2,000 μ2 of liver were taken of each specimen. The number of background grains in an adjacent area of equal size was determined for each specimen.

Morphology of hyaluronidase-sensitive cell coats as seen in the SEM after freeze-drying

1983 ◽  
Vol 62 (1) ◽  
pp. 371-383 ◽  
Author(s):  
J.B. Bard ◽  
W.H. McBride ◽  
A.R. Ross
Keyword(s):  
Exclusion Zone ◽  
Human Embryonic Lung ◽  
Electron Microscopic ◽  
Degree Of Hydration ◽  
Carbon Particles ◽  
Yield Information ◽  
Freeze Dried ◽  
Murine Fibrosarcoma

Many adherent cells in vitro are surrounded by a transparent exclusion zone or halo, several micrometers thick, which red blood cells, bacteria and carbon particles cannot penetrate. This halo is rapidly and specifically removed by hyaluronidase and its high degree of hydration is demonstrated by the fact that, although fixation does not eliminate the halo, solvent dehydration does. This latter observation means that the halo cannot be visualized by conventional electron microscopic techniques. We report here that the exclusion-zone material can, however, be seen in the scanning electron microscope if cells are fixed and frozen rapidly and then freeze-dried. Many cells in cultures from a murine fibrosarcoma or from human embryonic lung treated in this way appear to be covered by a matrix that obscures the microvilli that are visible on critical-point-dried or hyaluronidase-treated, freeze-dried cells. Only where the coat is, for some reason, missing can microvilli be seen on freeze-dried cells. The coat structure varies from amorphous to an assembly of fine fibres approximately 100 nm in diameter and its appearance is very similar to that of small drops of hyaluronic acid (10(−5) micrograms ml-1) treated in the same way. Halo material is fragile and detaches itself from the cell surface within an hour of fixation. These observations suggest that the halo phenomenon reflects only the production of extracellular matrix and its turnover. The fragility of the haloes implies that, if they do exist in vivo, they are unlikely to play any structural role. The results suggest that the technique will yield information on other highly hydrated, unstable structures.

Effect of osmium vapor on calcium loss during sectioning of freeze-dried, embedded tissue

1986 ◽  
Vol 44 ◽  
pp. 258-259
Author(s):  
L. J. McGuffee ◽  
S. A. Little
Keyword(s):  
Smooth Muscle ◽  
Freeze Drying ◽  
Osmium Tetroxide ◽  
Electron Microscopic ◽  
Electron Microscopic Autoradiography ◽  
Freeze Dried ◽  
Quick Freezing ◽  
Calcium Loss ◽  
Cellular Organelles

Our laboratory has been using electron microscopic autoradiography to localize 45Ca in smooth muscle. We prepare the tissue for these studies by quick freezing against a copper mirror, freeze-drying at low temperature, exposing the dry tissue to osmium tetroxide vapors in vacuo, and infiltrating and embedding in Spurr resin.Two requirements must be met before one can examine the distribution of a soluble ion, such as calcium, using this, or any morphological technique. First, morphological perservation of the tissue must be sufficient to identify cellular organelles and membranes. This requirement can be met in smooth muscle by using freeze-dried Spurr embedded tissue. Second, the distribution of calcium must be representative of the in vivo distribution.

scholarly journals Overview about the localization of nanoparticles in tissue and cellular context by different imaging techniques

2015 ◽  
Vol 6 ◽  
pp. 263-280 ◽  
Author(s):  
Anja Ostrowski ◽  
Daniel Nordmeyer ◽  
Alexander Boreham ◽  
Cornelia Holzhausen ◽  
Lars Mundhenk ◽  
...  
Keyword(s):  
Imaging Techniques ◽  
Optical Resolution ◽  
Fluorescence Lifetime Imaging ◽  
Structured Illumination ◽  
Electron Microscopic ◽  
Electron Microscopic Autoradiography ◽  
Technical Requirements ◽  
Tissue Responses ◽  
Recent Developments

The increasing interest and recent developments in nanotechnology pose previously unparalleled challenges in understanding the effects of nanoparticles on living tissues. Despite significant progress in in vitro cell and tissue culture technologies, observations on particle distribution and tissue responses in whole organisms are still indispensable. In addition to a thorough understanding of complex tissue responses which is the domain of expert pathologists, the localization of particles at their sites of interaction with living structures is essential to complete the picture. In this review we will describe and compare different imaging techniques for localizing inorganic as well as organic nanoparticles in tissues, cells and subcellular compartments. The visualization techniques include well-established methods, such as standard light, fluorescence, transmission electron and scanning electron microscopy as well as more recent developments, such as light and electron microscopic autoradiography, fluorescence lifetime imaging, spectral imaging and linear unmixing, superresolution structured illumination, Raman microspectroscopy and X-ray microscopy. Importantly, all methodologies described allow for the simultaneous visualization of nanoparticles and evaluation of cell and tissue changes that are of prime interest for toxicopathologic studies. However, the different approaches vary in terms of applicability for specific particles, sensitivity, optical resolution, technical requirements and thus availability, and effects of labeling on particle properties. Specific bottle necks of each technology are discussed in detail. Interpretation of particle localization data from any of these techniques should therefore respect their specific merits and limitations as no single approach combines all desired properties.

scholarly journals Ultrastructural discrimination of lipid droplets and vesicles in atherosclerosis: value of osmium-thiocarbohydrazide-osmium and tannic acid-paraphenylenediamine techniques.

1988 ◽  
Vol 36 (10) ◽  
pp. 1319-1328 ◽  
Author(s):  
J R Guyton ◽  
K F Klemp
Keyword(s):  
Tannic Acid ◽  
Lipid Droplets ◽  
Lipid Vesicles ◽  
Processing Technique ◽  
Unesterified Cholesterol ◽  
Electron Microscopic ◽  
En Bloc ◽  
Arterial Tissue ◽  
Microscopic Studies ◽  
Complementary Techniques

Electron microscopy of atherosclerotic arterial tissue commonly fails to distinguish lipid vesicles from droplets, especially when these are found in the extracellular space. The distinction is important, because vesicular or membranous lipid is composed of phospholipid and unesterified cholesterol, whereas neutral lipid in droplet form implies the presence of cholesteryl ester in atherosclerosis. A new procedure with sequential tannic acid and p-phenylenediamine treatments of osmicated tissue (TA-PDA) allows reliable ultrastructural discrimination of lipid vesicles and droplets. The multilamellar character of many vesicles is revealed. Extracellular droplets are found to possess many surface pits associated with membranous blebs. Pitting of droplets is especially evident after the use of an alternative tissue processing technique, the osmium-thiocarbohydrazide-osmium (OTO) sequence applied en bloc. The two complementary techniques will prove useful for electron microscopic studies of atherosclerotic and other lipid-rich tissues.

Cellular distribution of 125I-endothelin-1 binding in rat kidney following in vivo labeling

1994 ◽  
Vol 267 (5) ◽  
pp. F845-F852 ◽  
Author(s):  
R. Dean ◽  
J. Zhuo ◽  
D. Alcorn ◽  
D. Casley ◽  
F. A. Mendelsohn
Keyword(s):  
Cellular Localization ◽  
Rat Kidney ◽  
Electron Microscopic Level ◽  
Electron Microscopic ◽  
Electron Microscopic Autoradiography ◽  
Endothelin 1 ◽  
Peritubular Capillaries ◽  
Fenestrated Endothelium

Endothelin-1 (ET-1) receptors have previously been demonstrated in the rat kidney by in vitro autoradiography and in cultured renal cell lines by radioreceptor assay, but the precise cellular localization of these receptors under in vivo conditions remains to be determined. We performed electron microscopic autoradiography on rat kidney following intravenous administration of 125I-labeled ET-1. In vivo autoradiographs revealed binding patterns identical to those previously demonstrated following in vitro labeling. Light microscopic autoradiography showed that silver grains occurred exclusively overlaying glomeruli and peritubular capillaries in the cortex, inner stripe of the outer medulla, and the inner medulla. At the electron microscopic level, ET-1 binding was specifically localized to the fenestrated endothelium of glomerular and peritubular capillaries, and to a lesser extent to the vasa recta. No significant grains were seen on mesangial or visceral epithelial cells; nor were any seen on the cells of proximal tubule, the thick and thin limbs of the loop of Henle, the medullary collecting ducts, and renal interstitial cells. These results indicate that the endothelial cells of glomerular and peritubular capillaries are the primary target for the circulating ET-1 in the rat kidney and suggest an autocrine and/or paracrine function of locally synthesized ET-1 in vivo in both physiological and pathophysiological states.

Labelling of human resting lymphocytes by continuous infusion of [3H]thymidine. I. Characterization of cytoplasmic label

1978 ◽  
Vol 33 (1) ◽  
pp. 351-362
Author(s):  
P. Schick ◽  
F. Trepel ◽  
K.H. Maisel ◽  
W. Past ◽  
I. Reisert ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Mammalian Cells ◽  
Turnover Time ◽  
Electron Microscopic ◽  
Electron Microscopic Autoradiography ◽  
Resting Lymphocytes ◽  
Cytoplasmic Labelling

After continuous 3H-TdR infusion in vivo or incubation with 3H-TdR in vitro human blood lymphocytes were examined by light-microscopic and electron-microscopic autoradiography. Using relatively long autoradiographic exposure times (50–300 days) not only nuclear but also cytoplasmic labelling was visualized, the cytoplasmic label being present in up to 96% of the cells. The cytoplasmic label was predominantly associated with the mitochondria and was removed from the cells nearly completely by treatment with DNase but not with RNase or cold perchloric acid. It is concluded that this cytoplasmic label mainly represents 3H-TdR incorporated into mitochondrial DNA which is continuously renewed in an average turnover time of 14 days or less. This value is compatible with a turnover time of 11 days for mitochondrial DNA in mammalian cells reported in the literature.

scholarly journals Block-staining tissues with potassium ferrocyanide-reduced osmium tetroxide and lead aspartate for electron microscopic radioautography.

1984 ◽  
Vol 32 (5) ◽  
pp. 552-554 ◽  
Author(s):  
B M Kopriwa
Keyword(s):  
Room Temperature ◽  
Osmium Tetroxide ◽  
Uranyl Acetate ◽  
Potassium Ferrocyanide ◽  
Electron Microscopic ◽  
En Bloc ◽  
Simple Method ◽  
Electron Microscopic Radioautography

In the hope of devising a method for prestaining tissues en bloc for electron microscopic radioautography, pieces of radioiodine-labeled liver were taken through various combinations of ferrocyanide-reduced osmium tetroxide, lead aspartate, and aqueous uranyl acetate at room temperature or at 60 degrees C. Following the tests, the method adopted for routine use was to block-stain tissues for 2 hr in potassium ferrocyanide-reduced osmium tetroxide at 4 degrees C followed by 1 hr in Walton's lead aspartate at room temperature. This simple method, which requires no manipulation before or after emulsion coating and development of the radioautographs, provides adequate contrast without inducing background fog or artifacts.

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