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.

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.

Ein Verfahren zur elektronenmikroskopischen Untersuchung von Gewebekulturen im Wuchsverband

1965 ◽  
Vol 20 (5) ◽  
pp. 477-476b ◽  
Author(s):  
K. D. Zang ◽  
S. Luh ◽  
H. Hager
Keyword(s):  
Brain Tumors ◽  
Human Brain ◽  
Microscopic Study ◽  
Electron Micrographs ◽  
Osmium Tetroxide ◽  
Tissue Cultures ◽  
Improved Method ◽  
Electron Microscopic ◽  
Cellophane Sheet ◽  
Sectioned Tissue

An improved method for the electron microscopic study of sectioned tissue cultures of human brain tumors is based on commercial perforated cellophane sheets upon which the cells are grown. Specimens are fixed with buffered osmium tetroxide, imbedded horizontally in Metacrylate or Vestopal without separating them from the cellophane sheet and the monolayer cut in sections of 500 A.Electron micrographs show a very good preservation of intracellular and intercellular structures.

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.

Further Observations on the Virus of Epizootic Diarrhea of Infant Mice. An Electron Microscopic Study

1967 ◽  
Vol 25 ◽  
pp. 110-111
Author(s):  
W. G. Banfield ◽  
G. Kasnic ◽  
J. H. Blackwell
Keyword(s):  
Electron Microscope ◽  
Infected Cell ◽  
Microscopic Study ◽  
Intestinal Epithelium ◽  
Ultrastructural Study ◽  
Osmium Tetroxide ◽  
Lead Citrate ◽  
Electron Microscopic ◽  
Virus Particles ◽  
Infected Cells

An ultrastructural study of the intestinal epithelium of mice infected with the agent of epizootic diarrhea of infant mice (EDIM virus) was first performed by Adams and Kraft. We have extended their observations and have found developmental forms of the virus and associated structures not reported by them.Three-day-old NLM strain mice were infected with EDIM virus and killed 48 to 168 hours later. Specimens of bowel were fixed in glutaraldehyde, post fixed in osmium tetroxide and embedded in epon. Sections were stained with uranyl magnesium acetate followed by lead citrate and examined in an updated RCA EMU-3F electron microscope.The cells containing virus particles (infected) are at the tips of the villi and occur throughout the intestine from duodenum through colon. All developmental forms of the virus are present from 48 to 168 hours after infection. Figure 1 is of cells without virus particles and figure 2 is of an infected cell. The nucleus and cytoplasm of the infected cells appear clearer than the cells without virus particles.

Localization of Gallium-67 in Peritoneal Cells by Electron Microscopic Autoradiography

1973 ◽  
Vol 31 ◽  
pp. 404-405
Author(s):  
D. C. Swartzendruber ◽  
Norma L. Idoyaga-Vargas
Keyword(s):  
Clinical Trials ◽  
Soft Tissue ◽  
Tumor Tissue ◽  
Soft Tissue Tumors ◽  
Clinical Usefulness ◽  
Electron Microscopic ◽  
Normal Cells ◽  
Electron Microscopic Autoradiography ◽  
Peritoneal Cells ◽  
Gallium 67

The radionuclide gallium-67 (67Ga) localizes preferentially but not specifically in many human and experimental soft-tissue tumors. Because of this localization, 67Ga is used in clinical trials to detect humar. cancers by external scintiscanning methods. However, the fact that 67Ga does not localize specifically in tumors requires for its eventual clinical usefulness a fuller understanding of the mechanisms that control its deposition in both malignant and normal cells. We have previously reported that 67Ga localizes in lysosomal-like bodies, notably, although not exclusively, in macrophages of the spocytaneous AKR thymoma. Further studies on the uptake of 67Ga by macrophages are needed to determine whether there are factors related to malignancy that might alter the localization of 67Ga in these cells and thus provide clues to discovering the mechanism of 67Ga localization in tumor tissue.

Cytoplasmic Invaginations in Trophoblasts and Epithelial Cells of Rat

1971 ◽  
Vol 29 ◽  
pp. 524-525
Author(s):  
Iracema M. Baccarini
Keyword(s):  
Osmium Tetroxide ◽  
Uranyl Acetate ◽  
Nuclear Inclusions ◽  
Mucous Cells ◽  
Electron Microscopic ◽  
Cervical Epithelium ◽  
Intact Membrane ◽  
Microscopic Studies ◽  
Abnormal Cells ◽  
Nuclear Features

Some morphological nuclear features (invaginations) in normal and abnormal cells have been described in several electron microscopic studies. They have been referred to by others as blebs, loops, pockets, sheets, bodies, nuclear inclusions and cytoplasmic invaginations. Identical appearing structures were found in cells of the uterine cervical epithelium, in trophoblasts of blastocysts and in trophoblasts of rat placenta.Methods. Uterine cervix (normal rats), rat placenta (9-10 days gestation) and blastocyst were placed in 3% glutarahdehyde for 3 hours. The tissue was washed in phosphate buffer for 24 hours, postfixed in 1%. buffered osmium tetroxide for 1-2 hours and embedded in epon araldite. Sections were double stained with uranyl acetate and lead citrate and viewed in E. M. Siemens 200.Observations. Nuclear invaginations were found in basal, parabasal and mucous cells of the cervix epithelium, in trophoblasts of blastocyst and in trophoblasts of placenta. An oval, round or elongated invagination contained heterogenously cytoplasm surrounded by a double intact membrane; usually several invaginations were found in the same nucleus.

Electron Microscopic Examination of a Transplantable Rat Mammary Carcinoma

1968 ◽  
Vol 26 ◽  
pp. 20-21
Author(s):  
S. Shirahama ◽  
G. C. Engle ◽  
R. M. Dutcher
Keyword(s):  
Electron Microscope ◽  
Osmium Tetroxide ◽  
Electron Microscopic Examination ◽  
Undifferentiated Carcinoma ◽  
Uranyl Acetate ◽  
Sprague Dawley ◽  
Electron Microscopic ◽  
North West ◽  
X Irradiation ◽  
Tissue Specimens

A transplantable carcinoma was established in North West Sprague Dawley (NWSD) rats by use of X-irradiation by Engle and Spencer. The tumor was passaged through 63 generations over a period of 32 months. The original tumor, an adenocarcinoma, changed into an undifferentiated carcinoma following the 19th transplant. The tumor grew well in NWSD rats of either sex at various ages. It was invariably fatal, causing death of the host within 15 to 35 days following transplantation.Tumor, thymus, spleen, and plasma from 7 rats receiving transplants of tumor at 3 to 9 weeks of age were examined with an electron microscope at intervals of 8, 15, 22 and 30 days after transplantation. Four normal control rats of the same age were also examined. The tissues were fixed in glutaraldehyde, postfixed in osmium tetroxide and embedded in Epon. The plasma was separated from heparanized blood and processed as previously described for the tissue specimens. Sections were stained with uranyl acetate followed by lead citrate and examined with an RCA EMU-3G electron microscope.

X-Ray Microanalysis of Nickel and Cobalt Intensified Peroxidase- Antiperoxidase For Verification of Reaction Sites

1985 ◽  
Vol 43 ◽  
pp. 468-469
Author(s):  
A. Angel ◽  
K. Miller ◽  
V. Seybold ◽  
R. Kriebel
Keyword(s):  
Reaction Mechanisms ◽  
Visual Discrimination ◽  
Osmium Tetroxide ◽  
Ultrastructural Level ◽  
Microscopic Level ◽  
Electron Microscopic Level ◽  
Electron Microscopic ◽  
X Ray ◽  
Insoluble Polymer ◽  
Cytochemical Reaction

Localization of specific substances at the ultrastructural level is dependent on the introduction of chemicals which will complex and impart an electron density at specific reaction sites. Peroxidase-antiperoxidase(PAP) methods have been successfully applied at the electron microscopic level. The PAP complex is localized by addition of its substrate, hydrogen peroxide and an electron donor, usually diaminobenzidine(DAB). On oxidation, DAB forms an insoluble polymer which is able to chelate with osmium tetroxide becoming electron dense. Since verification of reactivity is visual, discrimination of reaction product from osmiophillic structures may be difficult. Recently, x-ray microanalysis has been applied to examine cytochemical reaction precipitates, their distribution in tissues, and to study cytochemical reaction mechanisms. For example, immunoreactive sites labelled with gold have been ascertained by means of x-ray microanalysis.

The Identification of Phospholipid Micelles in Glutaraldehyde-Urea Embedded Tissue

1975 ◽  
Vol 33 ◽  
pp. 494-495
Author(s):  
Daniel C. Pease
Keyword(s):  
Electron Micrographs ◽  
Lung Tissue ◽  
Osmium Tetroxide ◽  
Uranyl Acetate ◽  
Type Ii ◽  
Lead Citrate ◽  
Phospholipid Micelles ◽  
Type Ii Pneumocytes ◽  
Ultrathin Sections ◽  
Polar Water

It is reasonable to think that phospholipid micelles should be visible and identifiable in electron micrographs of ultrathin sections if only they can be preserved throughout the embedding process. The development of highly polar, water-containing, aminoplastic embedments has made this a likely possibility. With this in mind, an investigation of the lecithin-secreting, Type II pneumocytes of the lung is underway.Initially it has been easiest to recognize phospholipid micelles in lung tissue fixed first with glutaraldehyde, and then secondarily exposed to osmium tetroxide. However, the latter is not a necessary concomitant for micellar preservation. Conventional uranyl acetate and lead citrate staining is finally applied. Importantly, though, the micelles have been most easily seen in tissue embedded in 507. glutaraldehyde polymerized with urea, as described in detail by D.C. Pease and R.G. Peterson (J. Ultra- struct. Res., 41, 133, 1972). When oriented appropriately, the micellar units are seen as tiny, bilayer plates.

TEM comparison of osmium vs osmium with potassium ferricyanide secondary fixatives and the impact of secondary fixative temperature on tissue preservation/contrast quality

1996 ◽  
Vol 54 ◽  
pp. 910-911
Author(s):  
J. W. Horn ◽  
B. J. Dovey-Hartman ◽  
V. P. Meador
Keyword(s):  
Osmium Tetroxide ◽  
Potassium Ferricyanide ◽  
Electron Microscopic ◽  
Transmission Electron ◽  
Microscopic Evaluation ◽  
Cacodylate Buffer ◽  
Transmission Electron Microscopic ◽  
Glutaraldehyde Solution ◽  
Temperature Impact ◽  
The Impact

Osmium tetroxide (OsO4) is a universally used secondary fixative for routine transmission electron microscopic evaluation of biological specimens. Use of OsO4 results in good ultrastructural preservation and electron density but several factors, such as concentration, length of exposure, and temperature, impact overall results. Potassium ferricyanide, an additive used primarily in combination with OsO4, has mainly been used to enhance the contrast of lipids, glycogen, cell membranes, and membranous organelles. The purpose of this project was to compare the secondary fixative solutions, OsO4 vs. OsO4 with potassium ferricyanide, and secondary fixative temperature for determining which combination gives optimal ultrastructural fixation and enhanced organelle staining/contrast.Fresh rat liver samples were diced to ∼1 mm3 blocks, placed into porous processing capsules/baskets, preserved in buffered 2% formaldehyde/2.5% glutaraldehyde solution, and rinsed with 0.12 M cacodylate buffer (pH 7.2). Tissue processing capsules were separated (3 capsules/secondary fixative.solution) and secondarily fixed (table) for 90 minutes. Tissues were buffer rinsed, dehydrated with ascending concentrations of ethanol solutions, infiltrated, and embedded in epoxy resin.

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