scholarly journals LOSS OF PROTEINS AND OTHER MACROMOLECULES DURING PREPARATION OF CELL CULTURES FOR HIGH RESOLUTION AUTORADIOGRAPHY QUANTITATION BY A MICROMETHOD

1970 ◽  
Vol 18 (8) ◽  
pp. 565-573 ◽  
Author(s):  
TAPANI VANHA-PERTTULA ◽  
PHILIP M. GRIMLEY
Keyword(s):  
Amino Acids ◽  
High Resolution ◽  
Cell Cultures ◽  
Protein Extraction ◽  
Electron Microscopic ◽  
Human Carcinoma ◽  
Electron Microscopic Autoradiography ◽  
Binding Effect ◽  
Aldehyde Fixation ◽  
High Resolution Autoradiography

A reproducible procedure was devised in order to measure the extraction of 3H-labeled cellular products during aldehyde fixation and subsequent processing for electron microscopic autoradiography. Human carcinoma cell monolayers were cultivated in combustible plastic wells, so that all label could be counted as 3H2O. Radioisotope extraction during individual steps of processing could be analyzed and separate groups of experiments were directly comparable. Initial aldehyde fixation and subsequent buffer washes caused the major loss of radiolabeled amino acids, but this never exceeded 15% under conventional conditions. Radioisotope losses were influenced by the relative duration of fixation and buffer washes, fixation temperature and fixative concentration. Formaldehyde and glutaraldehyde both produced a nonspecific, time-related binding effect when 3H-labeled amino acids were introduced along with the fixative. Less significant nonspecific binding was observed when 3H-mannose, 3H-uridine or 3H-thymidine was added. Extraction of radioisotopes during formaldehyde fixation of cell cultures labeled with protein precursors was consistently greater than during glutaraldehyde fixation. Differences were less marked with the other precursors. Evaluation of the total protein extraction is complex, since the net losses observed were apparently the sum of precursor extraction, nonspecific amino acid binding and real molecular extraction. The implications for quantitative interpretative interpretation of high resolution autoradiography must be considered.

scholarly journals HIGH-RESOLUTION AUTORADIOGRAPHY

1962 ◽  
Vol 15 (2) ◽  
pp. 173-188 ◽  
Author(s):  
Lucien G. Caro ◽  
Robert P. van Tubergen
Keyword(s):  
High Resolution ◽  
Silver Halide ◽  
Electron Microscopic ◽  
Simple Method ◽  
Electron Microscopic Autoradiography ◽  
Fine Grain ◽  
High Resolution Autoradiography ◽  
Silver Halide Crystals ◽  
Subcellular Level ◽  
Physical Developer

Methods used in obtaining high resolution in autoradiography, with special emphasis on the technique of electron microscopic autoradiography, are described, together with control experiments designed to establish the optimum conditions or procedures. On the basis of these experiments the emulsion selected was Ilford L-4, with a crystal size slightly larger than 0.1 micron. It is applied to the specimen in the form of a gelled film consisting of a monolayer of silver halide crystals. Background, when present, can be eradicated by a simple method. The preparations can be stored, in presence of a drying agent, at room temperature or in a refrigerator. Photographic development is done in Microdol, or in a special fine grain "physical" developer. For examination in the electron microscope the sections are stained with uranyl or lead stains. These methods give a good localization of the label, at the subcellular level, and good reproducibility in relative grain counts.

Mise en évidence du rôle, dans la régénération des Amphibiens, d'une glycoprotéine sécrétée par la cape apicale: étude cytochimique et autoradiographique en microscopie électronique

Development ◽  
1974 ◽  
Vol 32 (1) ◽  
pp. 133-145
Author(s):  
Par Claude Chapron
Keyword(s):  
High Resolution ◽  
Epithelial Cells ◽  
Limb Regeneration ◽  
Target Cells ◽  
Silver Particles ◽  
Electron Microscopic ◽  
Microscopic Studies ◽  
High Resolution Autoradiography

Evidence for the role of an apical cap glycoprotein in amphibian regeneration: cytochemical and autoradiographic electron-microscopic studies Early during limb regeneration in the newt, an ectodermal apical cap covering a mesodermal blastema is formed. High-resolution autoradiography of these tissues has been carried out after incorporation of [3H]fucose, which is a precursor of glycoproteins. Autoradiography shows that silver particles are located at first on epithelial cells, then on mesenchymatous cells. This observation is consistent with a hypothesis in which the apical cap would elaborate a glycoprotein acting on the blastema. Substructural autoradiography and cytochemistry also show the importance of cellular surfaces for both cells producing glycoprotein and those which are target cells.

scholarly journals HIGH RESOLUTION AUTORADIOGRAPHY WITH STRIPPING FILM

1971 ◽  
Vol 19 (5) ◽  
pp. 304-309 ◽  
Author(s):  
JOSEPH R. WILLIAMSON ◽  
HANK VAN DEN BOSCH
Keyword(s):  
High Resolution ◽  
Mechanical Device ◽  
Electron Microscopic ◽  
High Quality ◽  
Simple Method ◽  
Thin Sections ◽  
Large Numbers ◽  
High Resolution Autoradiography ◽  
Potential Doubling

A simple method is described for the preparation and use of stripping film of high quality for electron microscopic applications of autoradiography. The advantages of the method are: ( a) uniform monolayers of emulsion are easily prepared with an inexpensive mechanical device and applied to thin sections on conventional grids; ( b) both sides of grids can be coated with emulsion, permitting a potential doubling of sensitivity with, theoretically, no loss of resolution; and ( c) large numbers of grids can be processed simultaneously, assuring identical conditions of development.

Electron Microscopic Autoradiography of Rabbit Thyroid Gland Following Calcitonin

1973 ◽  
Vol 31 ◽  
pp. 676-677
Author(s):  
A. P. Lupulescu ◽  
F. C. Stebner
Keyword(s):  
Salmon Calcitonin ◽  
Medical Research Council ◽  
Electron Microscopic ◽  
Electron Microscopic Autoradiography ◽  
Thyroid Glands ◽  
Thyroid Follicular Cells ◽  
Term Administration ◽  
High Resolution Autoradiography ◽  
And Control

In a previous paper we observed that long-term administration of salmon calcitonin (SCT) exerts a goitrogenic effect on rabbit thyroid glands. In order to explain if this is a direct effect on thyroid follicular cells or mediated through other factors, we studied the cellular and intracellular radioiodine (125I) distribution by using high resolution autoradiography.For electron microscopic autoradiography, five adult rabbits were injected i.m. three times weekly with salmon calcitonin (Armour Pharmaceutical Company), total dose of 800 MRCU (Medical Research Council Units) per rabbit. Another three rabbits served as controls. Both calcitonin-treated and control rabbits received 500 μc radioiodine (125I) intraperitoneally (each rabbit) and thyroid lobes were removed 1½ hours after isotope and calcitonin administration.

scholarly journals Back to back autoradiography: a method for ultrastructural analysis of light microscopic autoradiographs.

1980 ◽  
Vol 28 (3) ◽  
pp. 276-278 ◽  
Author(s):  
N B Kaplan ◽  
L J Smith ◽  
J S Brody
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
High Resolution ◽  
Ultrastructural Analysis ◽  
Thick Section ◽  
Electron Microscopic ◽  
Electron Microscopic Autoradiography ◽  
Glass Slides

A method for studying radiolabeled cells is described that combines the simplicity of light microscopic autoradiography with the high resolution of electron microscopy. Serial thin (600 A) and thick (1 micron) sections are placed on Formvar-coated slot grids and glass slides, respectively. Labeled cells are visualized on the thick section by autoradiography and may then be studied in the electron microscope by locating the corresponding fields on the grid. This technique permits accurate ultrastructural identification and analysis of radiolabeled cells, yet avoids the need for electron microscopic autoradiography.

Sign in / Sign up

Export Citation Format

Share Document