scholarly journals An improved method for freeze-fracture radioautography of tissues and cells, as applied to duodenal epithelium and thymic lymphocytes.

1984 ◽  
Vol 32 (1) ◽  
pp. 17-29 ◽  
Author(s):  
F W Kan ◽  
B M Kopriwa ◽  
C P Leblond
Keyword(s):  
Freeze Fracture ◽  
Electron Microscopic Examination ◽  
Improved Method ◽  
Electron Microscopic ◽  
Carbon Replica ◽  
Radiation Sources ◽  
Freeze Dried ◽  
Duodenal Epithelium ◽  
Tissue Specimens ◽  
Silver Grains

An improved method has been devised for the localization of radioactive substances to either one of the leaflets of cellular membranes. After tissue specimens are freeze-fractured and covered with a platinum-carbon replica, they are freeze-dried to allow coating with radioautographic emulsion at room temperature. After exposure at 4 degrees C and development, the emulsion is protected by layers of carbon and grease before the tissue underlying the replica is dissolved in sodium hypochlorite. The grease is removed in Freon 14 and the replica with its emulsion cover is mounted on a specimen grid for electron microscopic examination. The accuracy of radioactivity localization was demonstrated using 3H-thymidine-labeled liver by finding silver grains over the same sites after freeze-fracture as after thin section radioautography. Tests with 3H-methacrylate revealed that the interposition of a platinum-carbon replica decreased the radioautographic reaction by over 80%; hence, the need for long exposure. Only 67% of the silver grains came from radiation sources located beyond the upper 0.05 micron of the specimen and, therefore, the emulsion could be affected by radiation sources located not only within membrane leaflets but also in nearby cytoplasm. Thus, when 3H-fucose was injected into rats to locate newly formed glycoproteins within intestinal epithelium membranes, some of the silver grains found over E and P faces might be produced by radiation coming from the adjacent cytoplasm. To localize label within membrane leaflets in the absence of radiation sources in the cytoplasm, lymphocyte suspensions were incubated with 3H-concanavalin A at 0 degrees C. The plasmalemma radioactivity was then restricted to the two membrane leaflets, with 87-93% of the silver grains on the E leaflet and 7-13% on the P leaflet. It appears that, under these conditions, the technique provides adequate localization of radioactivity to the leaflets of the cell membrane.

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.

Quick-Freezing With High Time Resolution Of Fast Physiological Events

1985 ◽  
Vol 43 ◽  
pp. 6-9
Author(s):  
Joachim R. Sommer
Keyword(s):  
Time Scale ◽  
Biological Material ◽  
Morphological Changes ◽  
Freeze Fracture ◽  
Time Interval ◽  
High Time Resolution ◽  
Electron Microscopic ◽  
Freeze Dried ◽  
Quick Freezing ◽  
Spatial Displacements

Quick-freezing methodology has made three major contributions to our ability to relate structure to function: 1. Quick-freezing, especially when followed by freeze-fracture, is suited to study the ultrastructure of unfixed biological material as close to the native state as can possibly be obtained. 2. Physiological events associated with morphological changes at the level of electron microscopic resolution can be stopped at any desired time interval and, thus, analysed against a known time scale and, 3. Microchemical measurements, e.g. of elemental concentrations and their spatial displacements, can be obtained by electron dispersive X-ray microanalysis from freeze-dried frozen sections of quick-frozen biological material. Indeed, it is now possible to investigate, simultaneously, anatomical, physiological and microchemical parameters in a single cell, with a precise time scale thrown in for good measure.In the following I shall describe the techniques that we employ to study the morphology of single intact skeletal muscle fibers of the frog at known time intervals following electrical stimulation. The methodology is the judicious extension, including some modifications, of procedures whose efficacy is mostly uncontroversial and a matter of scientific record.

Freeze-fracture architecture and polypeptide composition of thylakoid membranes from euploid Ricinus cells

1981 ◽  
Vol 52 (1) ◽  
pp. 167-181
Author(s):  
M.P. Timko ◽  
R.E. Triemer ◽  
A.C. Vasconcelos
Keyword(s):  
Ricinus Communis ◽  
Relative Proportion ◽  
Freeze Fracture ◽  
Nuclear Genome ◽  
Electron Microscopic Examination ◽  
Thylakoid Membranes ◽  
Polypeptide Composition ◽  
Electron Microscopic ◽  
Size Category ◽  
Ploidy Levels

The freeze-fracture architecture and polypeptide composition of thylakoid membranes of euploid cells of Ricinus communis L. were examined. Electron microscopic examination of the chloroplasts of 1N 2N and 4N cells revealed little variation in the size of chloroplasts, lamellar structure and internal organization of plastids, despite increases in plastid numbers per cell observed to accompany the increase in nuclear ploidy. Thylakoid membranes from euploid cells were also similar in their freeze-fracture morphology. Two basic types of intramembranous particles were observed on the fracture faces of thylakoid membranes of euploid cells. The endoplasmic fracture (EF) face of experimentally unstacked thylakoid membranes of 1N, 2N and 4N cells contain 2 size categories of particles (115-121 A and 164–166 A), whereas the protoplasmic fracture (PF) face of these membranes contain a single size category of particles (85-88 A). The distribution and size of the EF- and PF-face particles were found to be similar among membranes from cells of the 3 ploidy levels. Analysis of the polypeptide composition of thylakoid membranes from 1N, 2N and 4N cells revealed no difference in the relative proportion of the constituent polypeptides of these membranes. The possible factors involved in the regulation of the development of thylakoid structure and composition in the presence of altered nuclear genome size are discussed.

scholarly journals Polyomavirus Encephalopathy in a Ducorps Cockatoo (Cacatua Ducorpsii) with Psittacine Beak and Feather Disease

1996 ◽  
Vol 8 (3) ◽  
pp. 291-295 ◽  
Author(s):  
Kenneth S. Latimer ◽  
Frank D. Niagro ◽  
W. L. Steffens ◽  
Branson W. Ritchie ◽  
Raymond P. Campagnoli
Keyword(s):  
In Situ Hybridization ◽  
Nucleic Acid ◽  
Neuronal Degeneration ◽  
Electron Microscopic Examination ◽  
Electron Microscopic ◽  
Histologic Evaluation ◽  
Tissue Specimens ◽  
Avian Polyomavirus ◽  
Formalin Fixed

Necropsy tissues were examined from an adult wild-caught Ducorps cockatoo (Cacatua ducorpsii) with progressive neurologic signs. Of the tissue specimens selected for histologic evaluation, only the brain contained rare amphophilic, glassy intranuclear inclusions within astrocytes and some neurons. Astrocyte and neuronal degeneration and necrosis also were observed. Scattered astrocytes, with and without discernable inclusions, contained avian polyomavirus (APV) nucleic acid, as determined by DNA in situ hybridization. In addition, endothelial cells and intravascular leukocytes contained psittacine beak and feather disease viral nucleic acid, as determined by DNA in situ hybridization, indicating dual viral infection. Electron microscopic examination of formalin-fixed brain tissue revealed typical intranuclear APV particles in some astrocytes. Encephalopathy ultimately was attributed to APV infection.

Rhodopsin Particles in the Photoreceptor Membrane of an Insect

1976 ◽  
Vol 31 (11-12) ◽  
pp. 763-763 ◽  
Author(s):  
C. Bruce Boschek ◽  
Kurt Hamdorf
Keyword(s):  
Microscopic Examination ◽  
Visual Pigment ◽  
Particle Density ◽  
Substantial Reduction ◽  
Freeze Fracture ◽  
Electron Microscopic Examination ◽  
Electron Microscopic ◽  
Deficient Diet ◽  
Photoreceptor Membrane ◽  
Fracture Electron

Abstract Rhodopsin, Insect Photoreceptor, Freeze Fracture Electron-microscopic examination of freeze-fractured fly retinae has revealed the presence of particles, 80 to 100 Å in diameter, on the photoreceptor membrane. Flies which were raised on a vitamin-A deficient diet show a substantial reduction in the density of such particles. The reduction in particle density is in agreement with the reduction in visual-pigment concentration as measured spectrophotometrically for these flies. These results suggest that the particles are identical with molecules of the visual pigment, rhodopsin.

A Freeze-Fracture Technique for Examining Human Stratum Corneum

1972 ◽  
Vol 30 ◽  
pp. 298-299
Author(s):  
Daniel P. Hannon
Keyword(s):  
Electron Microscopy ◽  
Sample Preparation ◽  
Stratum Corneum ◽  
Microscopic Examination ◽  
Freeze Fracture ◽  
Electron Microscopic Examination ◽  
Electron Microscopic ◽  
Human Stratum Corneum ◽  
Freeze Fracture Electron Microscopy ◽  
Fracture Electron

Human stratum corneum is a difficult tissue to process for electron microscopic examination. Most of the difficulty is due to the inability of the chemical fixative to adequately preserve all of the constituents of this tissue. As a result, during sectioning, breaks occur between cells creating voids which preclude any studies of these areas. Also, because the fixative is usually in an aqueous medium, studies of hydrated or dehydrated states are impossible. However, these problems can be overcome by using an alternative method of sample preparation, freeze-fracture electron microscopy.Samples of human skin both untreated and soaked for 20 minutes in 20% glycerin were cut into strips 1 mm wide by 2 mm long and placed in specially made copper discs with a 1 mm deep central cup. Freezing was accomplished by immersion in liquid Freon 22.

scholarly journals Paranasal Meningioma in the Dog: A Clinicopathologic Study of Ten Cases

1986 ◽  
Vol 23 (4) ◽  
pp. 362-368 ◽  
Author(s):  
A. K. Patnaik ◽  
P. H. Lieberman ◽  
R. A. Erlandson ◽  
E. Shaker ◽  
A. I. Hurvitz
Keyword(s):  
Nasal Cavity ◽  
Microscopic Examination ◽  
Electron Microscopic Examination ◽  
Nasal Discharge ◽  
Cranial Cavity ◽  
Electron Microscopic ◽  
Clinicopathologic Study ◽  
Tissue Specimens ◽  
Histologic Types ◽  
The Brain

Paranasal meningiomas were diagnosed in ten dogs based on gross and light microscopic examinations of tissue specimens, and, in one case, electron microscopic examination. Seven of ten dogs were female (average age was 13 years). Most dogs (7/10) had seizures on examination. Two dogs with meningioma located in the nasal cavity had nasal discharge, and one had epistaxis. Tumors originated in the nasoparanasal region (eight) and frontal region of the cranial cavity (two). The histologic types of meningioma included psammomatous (two), transitional (three), meningotheliomatous (two), fibroblastic (two), and angioblastic (one). Tumors were malignant and extended to the brain in eight cases. These tumors differed from intracranial meningiomas mainly in their more anaplastic nature and aggressive behavior.

scholarly journals Monolayer freeze-fracture autoradiography: quantitative analysis of the transmembrane distribution of radioiodinated concanavalin A.

1982 ◽  
Vol 93 (1) ◽  
pp. 155-163 ◽  
Author(s):  
K A Fisher
Keyword(s):  
Concanavalin A ◽  
Biological Membrane ◽  
Freeze Fracture ◽  
Photographic Emulsion ◽  
Con A ◽  
Cell Monolayers ◽  
New Information ◽  
Freeze Dried ◽  
Iodine 125 ◽  
Silver Grains

The technique of monolayer freeze-fracture autoradiography (MONOFARG) has been developed and the principles, quantitation, and application of the method are described. Cell monolayers attached to polylysine-treated glass were freeze-fractured, shadowed, and coated with dry, Parlodion-supported Ilford L4 photographic emulsion at room temperature. Quantitative aspects of MONOFARG were examined using radioiodinated test systems. Background was routinely less than 2.5 X 10(-4) grains/microns 2/day, the highest overall efficiency was between 25% and 45%, and grain density and efficiency were dependent on radiation dose for iodine-125 and D-19 development. Corrected grain densities were linearly proportional to iodine-125 concentration. The method was applied to an examination of the transmembrane distribution of radioiodinated and fluoresceinated concanavalin A (125I-FITC-Con-A). Human erythrocytes were labeled, column-purified, freeze-dried or freeze-fractured, autoradiographed, and examined by electron microscopy. The number of silver grains per square micrometer of unsplit single membrane was essentially identical to that of split extracellular membrane "halves." These data demonstrate that 125I-FITC-Con-A partitions exclusively with the extracellular "half" of the membrane upon freeze-fracturing and can be used as a quantitative marker for the fraction of extracellular split membrane "halves." This method should be able to provide new information about certain transmembrane properties of biological membrane molecules and probes, as well as about the process of freeze-fracture per se.

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