scholarly journals CYTOCHEMICAL LOCALIZATION OF NUCLEIC ACIDS BY AN ACRIFLAVINE-PHOSPHOTUNGSTATE COMPLEX FOR FLUORESCENCE MICROSCOPY AND ELECTRON MICROSCOPY

1970 ◽  
Vol 18 (9) ◽  
pp. 609-627 ◽  
Author(s):  
VICTORIA CHAN-CURTIS ◽  
W. DUANE BELT ◽  
CHARLES T. LADOULIS
Keyword(s):  
Electron Microscopy ◽  
Fluorescence Microscopy ◽  
Nucleic Acids ◽  
Phosphotungstic Acid ◽  
Phosphomolybdic Acid ◽  
Testicular Tissue ◽  
Aqueous System ◽  
Single Step ◽  
Electron Microscopic ◽  
Cytochemical Localization

An acriflavine-phosphotungstate complex (A-PTA) was synthesized from a reaction between acriflavine and phosphotungstic acid, with the former in excess. The purified product is fluorescent and electron-dense, and was studied for its use as a cytochemical reagent for fluorescence microscopy and electron microscopy. A selectivity for nucleic acids and sulfated cerebroside esters that excludes sulfated glycoaminoglycans is proposed. The reaction for fluorescence microscopy is rapid, sensitive and reproducible. The electron-dense A-PTA complex localized nucleic acids for electron microscopy. Known sites of DNA and RNA in cells of pancreatic, cerebellar and testicular tissue have been demonstrated with the reaction. The reaction is administered as a single step, interposed in the electron microscopic preparative procedure, and can be used in either an alcoholic or an aqueous system. The alcoholic A-PTA reaction produced adequate contrast on short duration (2 hr); the aqueous block reaction was found most satisfactory when prolonged to 48 hr. Uranyl acetate, phosphomolybdic acid and phosphotungstic acid can be used as supplementary stains in addition to the A-PTA reaction. Under these conditions, DNP and RNP sites were demonstrated. The A-PTA complex is a prototype of the coupled dye-metal histochemical reagents, and its selectivity for nucleic acids represents a new approach in cytochemistry. Its reactions can be investigated by fluorescence microscopy and electron microscopy, and in solution. Other dye-metal reagents can probably be formulated on a similar design as detection systems for lipids, polysaccharide or specific groups in proteins.

mRNA localization by Electron microscopic in situ hybridization

1991 ◽  
Vol 49 ◽  
pp. 430-431
Author(s):  
J. A. Pollock ◽  
M. Martone ◽  
T. Deerinck ◽  
M. H. Ellisman
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Nucleic Acids ◽  
Recombinant Dna ◽  
Light And Electron Microscopy ◽  
Electron Microscopic ◽  
High Voltage Electron Microscopy ◽  
Functional Sites ◽  
Voltage Electron

Localization of specific proteins in cells by both light and electron microscopy has been facilitate by the availability of antibodies that recognize unique features of these proteins. High resolution localization studies conducted over the last 25 years have allowed biologists to study the synthesis, translocation and ultimate functional sites for many important classes of proteins. Recently, recombinant DNA techniques in molecular biology have allowed the production of specific probes for localization of nucleic acids by “in situ” hybridization. The availability of these probes potentially opens a new set of questions to experimental investigation regarding the subcellular distribution of specific DNA's and RNA's. Nucleic acids have a much lower “copy number” per cell than a typical protein, ranging from one copy to perhaps several thousand. Therefore, sensitive, high resolution techniques are required. There are several reasons why Intermediate Voltage Electron Microscopy (IVEM) and High Voltage Electron Microscopy (HVEM) are most useful for localization of nucleic acids in situ.

scholarly journals Correlating Fluorescence Microscopy with Electron Microscopy

2004 ◽  
Vol 12 (1) ◽  
pp. 3-7
Author(s):  
Stephen W. Carmichael ◽  
Jon Charlesworth
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Fluorescence Microscopy ◽  
Fluorescent Probes ◽  
Cell Biology ◽  
Microscopic Image ◽  
Electron Microscopic ◽  
Transmission Electron ◽  
Electron Microscopic Image ◽  
Auto Fluorescence

The use of fluorescent probes is becoming more and more common in cell biology. It would be useful if we were able to correlate a fluorescent structure with an electron microscopic image. The ability to definitively identify a fluorescent organelle would be very valuable. Recently, Ying Ren, Michael Kruhlak, and David Bazett-Jones devised a clever technique to correlate a structure visualized in the light microscope, even a fluorescing cell, with transmission electron microscopy (TEM).Two keys to the technique of Ren et al are the use of grids (as used in the TEM) with widely spaced grid bars and the use of Quetol as the embedding resin. The grids allow for cells to be identified between the grid bars, and in turn the bars are used to keep the cell of interest in register throughout the processing for TEM. Quetol resin was used for embedding because of its low auto fluorescence and sectioning properties. The resin also becomes soft and can be cut and easily peeled from glass coverslips when heated to 70°C.

scholarly journals Immunogold probes for electron microscopy: evaluation of staining by fluorescence microscopy.

1985 ◽  
Vol 33 (10) ◽  
pp. 995-1000 ◽  
Author(s):  
R B Alexander ◽  
W B Isaacs ◽  
E R Barrack
Keyword(s):  
Electron Microscopy ◽  
Fluorescence Microscopy ◽  
Intermediate Filaments ◽  
Colloidal Gold ◽  
Microscopic Level ◽  
Secondary Antibody ◽  
Light Microscopic Level ◽  
Electron Microscopic ◽  
Cell Monolayers ◽  
Adenocarcinoma Cells

A method is presented whereby the staining of intracellular structures with immunogold probes for electron microscopy can be evaluated at the light microscopic level. Methanol-fixed monolayers of cultured Dunning R-3327-H rat prostatic adenocarcinoma cells were stained for cytokeratins using a two-step immunogold technique consisting of primary anti-keratin antibody followed by gold-labeled secondary antibody. Bound immunogold probe was then visualized with a fluorescent tertiary anti-immunogold probe antibody. Fluorescence microscopy of the whole cell monolayers showed a typical keratin cytoskeleton. The extra staining step did not interfere with subsequent fixation, embedding, and sectioning for electron microscopy, which showed cytoplasmic intermediate filaments decorated with colloidal gold. Using this method, it should be possible to manipulate parameters critical to staining with immunogold probes and to evaluate the labeling without necessitating repeated time-consuming electron microscopic processing. The method also provides a useful correlation between the light microscopic and ultrastructural labeling patterns of immunogold probes.

scholarly journals A NEW ELECTRON-DENSE STAIN FOR ELASTIC TISSUE

1970 ◽  
Vol 18 (10) ◽  
pp. 697-708 ◽  
Author(s):  
ERNEST N. ALBERT ◽  
EVERLY FLEISCHER
Keyword(s):  
Electron Microscopy ◽  
Heavy Metals ◽  
Fluorescence Microscopy ◽  
Electron Density ◽  
The Body ◽  
Elastic Tissue ◽  
Electron Microscopic ◽  
Specific Staining ◽  
Microscopic Studies ◽  
Specific Affinity

Tetraphenylporphine sulfonate has been shown to have a specific affinity for elastic tissues of the body in fluorescence microscopy. However, tetraphenylporphine sulfonate does not impart electron density to this tissue and thus is not suitable for electron microscopic studies. Therefore, tetraphenylporphine sulfonate was complexed with various heavy metals in order to use it as a specific stain for elastic tissues in electron microscopy. The silver and gold metallic complexes gave the most consistent and specific staining reactions. These compounds were prepared in this laboratory. The synthesis and staining procedures are described in detail.

A Comparison of Light, Fluorescence and Electron Microscopic Observations in Assessing the SO2 Injury of Lichens Under Different Moisture Conditions

1989 ◽  
Vol 21 (2) ◽  
pp. 119-134 ◽  
Author(s):  
T. Holopainen ◽  
M. Kauppi
Keyword(s):  
Electron Microscopy ◽  
Fluorescence Microscopy ◽  
Light Microscopy ◽  
Lichen Species ◽  
High Humidity ◽  
Electron Microscopic ◽  
Fumigation Chamber ◽  
Low Humidity ◽  
Light Fluorescence ◽  
Moisture Conditions

AbstractThree lichen species, Hypogymnia physodes, Bryoria capillaris and Peltigera canina were fumigated with 215 μig m−3 of SO2 (6 h per day, 5 days per week) in exposure chambers under three different moisture conditions. The treatments were (1) low humidity (40–50° r.h. continuously), (2) high humidity (80–90° r.h. continuously) and (3) high humidity + water (80–90° r.h. + daily moistening). The value of light microscopy, fluorescence microscopy and electron microscopy in assessing the SO, injury in the lichens was compared. The three lichen species studied were all damaged by this level of SO, fumigation, and injury increased with increased duration of exposure. Electron microscopy was the most sensitive method of revealing cellular injuries after 5 days of exposure in all the species studied. Fluorescence microscopy revealed changes in the colour and intensity of fluorescence after 10 days of exposure in the two epiphytic species, but no clear changes in the cyanobacterial cells of P. canina could be detected by this method. Light microscopy revealed plasmolysis of the algal cells in the two epiphytic species after 10–15 days of fumigation. All the species studied were clearly more sensitive to SO2 when cultured in air of high humidity, although slight cellular injuries also occurred in the dry air treatment (40–50° r.h.). Additional daily moistening tended to protect against injury in the lichens cultured at high humidity. In the present continuous flow fumigation chamber P. canina proved to be sensitive to SO2 in regard to ultrastructural injuries that could be observed in both the cyanobacterial and fungal cells.

scholarly journals An electron microscopic histochemical and analytical X-ray microprobe study of calcification in Bruch's membrane from human eyes.

1981 ◽  
Vol 29 (5) ◽  
pp. 601-608 ◽  
Author(s):  
W L Davis ◽  
R G Jones ◽  
H K Hagler
Keyword(s):  
Electron Microscopy ◽  
Microprobe Analysis ◽  
Electron Microprobe Analysis ◽  
Phosphotungstic Acid ◽  
Electron Microscopic ◽  
X Ray ◽  
Transmission Electron ◽  
Dense Deposits ◽  
Calcium And Phosphorus ◽  
Human Eyes

Transmission electron microscopy, electron microprobe analysis, high temperature microincineration, and electron microscopic histochemical procedures were used to study the electron-dense deposits characteristic of the macular aspect of aged human eyes. These inorganic deposits were rich in calcium and phosphorus and selectively removed by flotation on formic acid. The amorphous decalcified masses showed a significant sulfur peak and were readily stained with acidic phosphotungstic acid. The latter observations are indicative of the presence of organic matrical proteoglycan. Such data may be a further indication that proteoglycans are retained at sites of calcification.

scholarly journals Ethidium bromide- and propidium iodide-PTA staining of nucleic acids at the electron microscopic level.

1989 ◽  
Vol 37 (7) ◽  
pp. 1161-1166 ◽  
Author(s):  
M Biggiogera ◽  
F F Biggiogera
Keyword(s):  
Nucleic Acids ◽  
Ethidium Bromide ◽  
Propidium Iodide ◽  
Phosphotungstic Acid ◽  
Microscopic Level ◽  
Electron Microscopic Level ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Dna And Rna ◽  
Fluorescent Markers

Ultra-thin sections of various tissues were stained with ethidium bromide or propidium iodide, two fluorescent markers widely used for quantitation of nucleic acids. The fluorochromes, tested at different concentrations, were then revealed by incubation of the sections with neutralized phosphotungstic acid. We showed that at the electron microscopic level only nucleic acid-containing structures are revealed. Chromatin, nucleolus, and ribosomes appear to be stained by the end-product of the reaction. Furthermore, controls with proteases and nucleases showed that the staining is related to the binding of the fluorochromes to DNA and RNA and to the subsequent detection of the dyes by neutralized PTA.

A New Electron Dense Stain For Elastin

1969 ◽  
Vol 27 ◽  
pp. 414-415
Author(s):  
Ernest N. Albert
Keyword(s):  
Electron Microscopy ◽  
Heavy Metals ◽  
Phosphotungstic Acid ◽  
Low Ph ◽  
The Body ◽  
Electron Microscopic ◽  
Virtual Absence ◽  
Microscopic Studies ◽  
Specific Affinity ◽  
General Protein

Only phosphotungstic acid (PTA) has been demonstrated to have a reasonably specific affinity for elastin in electron microscopy. However phosphotungstic acid is effective as a specific stain for elastin only at very acid pH. If the pH is increased, PTA is no more a specific stain for elastin, instead it acts as a general protein stain. Therefore at low pH only elastin is visible and one is unable to study the morphological relationships of the elastic components to their surrounding structures due to the virtual absence of contrast in them.Tetraphenylporphine sulfonate (TPPS) has, been shown to have a specific, affinity for elastic tissues of the body in fluorescence microscopy. However TPPS does not impart electron density to this tissue and thus is not suitable for electron microscopic studies. Therefore tetraphenylporphine sulfonate was complexed with various heavy metals in order to use it as a specific stain for elastin in electron microscopy.

Localization of immunolabels by electron microscopy and image averaging

1983 ◽  
Vol 41 ◽  
pp. 282-283
Author(s):  
J. Frank ◽  
P.-Y. Sizaret ◽  
A. Verschoor ◽  
J. Lamy
Keyword(s):  
Electron Microscopy ◽  
Single Particle ◽  
Attachment Site ◽  
Uranyl Acetate ◽  
Limulus Polyphemus ◽  
Resolution Limit ◽  
Electron Microscopic ◽  
Image Averaging ◽  
Top View ◽  
Better Than

The accuracy with which the attachment site of immunolabels bound to macromolecules may be localized in electron microscopic images can be considerably improved by using single particle averaging. The example studied in this work showed that the accuracy may be better than the resolution limit imposed by negative staining (∽2nm).The structure used for this demonstration was a halfmolecule of Limulus polyphemus (LP) hemocyanin, consisting of 24 subunits grouped into four hexamers. The top view of this structure was previously studied by image averaging and correspondence analysis. It was found to vary according to the flip or flop position of the molecule, and to the stain imbalance between diagonally opposed hexamers (“rocking effect”). These findings have recently been incorporated into a model of the full 8 × 6 molecule.LP hemocyanin contains eight different polypeptides, and antibodies specific for one, LP II, were used. Uranyl acetate was used as stain. A total of 58 molecule images (29 unlabelled, 29 labelled with antl-LPII Fab) showing the top view were digitized in the microdensitometer with a sampling distance of 50μ corresponding to 6.25nm.

Immunocytochemistry. A Review of Methodology for Electron Microscopic Applicaiton

1974 ◽  
Vol 32 ◽  
pp. 328-329
Author(s):  
Joseph E. Mazurkiewicz
Keyword(s):  
Electron Microscopy ◽  
Light Microscopy ◽  
Ultrastructural Level ◽  
Electron Microscopic ◽  
Biological Interest ◽  
Investigative Approach ◽  
Immunologic Activity ◽  
Dense Label

Immunocytochemistry is a powerful investigative approach in which one of the most exacting examples of specificity, that of the reaction of an antibody with its antigen, isused to localize tissue and cell specific molecules in situ. Following the introduction of fluorescent labeled antibodies in T950, a large number of molecules of biological interest had been studied with light microscopy, especially antigens involved in the pathogenesis of some diseases. However, with advances in electron microscopy, newer methods were needed which could reveal these reactions at the ultrastructural level. An electron dense label that could be coupled to an antibody without the loss of immunologic activity was desired.

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