Reconstruction of Two-Dimensional Projections of GP 32*I

1981 ◽  
Vol 39 ◽  
pp. 38-39
Author(s):  
H.A. Cohen ◽  
W. Chiu ◽  
J. Hosoda
Keyword(s):  
Electron Microscopy ◽  
Molecular Weight ◽  
Uranyl Acetate ◽  
Distilled Water ◽  
Acetate Solution ◽  
Two Dimensional ◽  
Parent Molecule ◽  
T4 Bacteriophage ◽  
Electron Imaging ◽  
Better Than

GP 32 (molecular weight 35000) is a T4 bacteriophage protein that destabilizes the DNA helix. The fragment GP32*I (77% of the total weight), which destabilizes helices better than does the parent molecule, crystallizes as platelets thin enough for electron diffraction and electron imaging. In this paper we discuss the structure of this protein as revealed in images reconstructed from stained and unstained crystals.Crystals were prepared as previously described. Crystals for electron microscopy were pelleted from the buffer suspension, washed in distilled water, and resuspended in 1% glucose. Two lambda droplets were placed on grids over freshly evaporated carbon, allowed to sit for five minutes, and then were drained. Stained crystals were prepared the same way, except that prior to draining the droplet, two lambda of aqueous 1% uranyl acetate solution were applied for 20 seconds. Micrographs were produced using less than 2 e/Å2 for unstained crystals or less than 8 e/Å2 for stained crystals.

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.

Fibrin-I Degradation Products with a Molecular Weight Higher than That of Fibrinogen

1973 ◽  
Vol 29 (01) ◽  
pp. 122-129 ◽  
Author(s):  
René von Hugo ◽  
Henner Graeff
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Degradation Products ◽  
Gel Filtration ◽  
Agarose Gel ◽  
Two Dimensional ◽  
Plasma Clot ◽  
Two Dimensional Gel Electrophoresis ◽  
Parent Molecule

SummaryThe observation of intravascular lysis of fibrin deposits and of fibrinogen derivatives with a molecular weight higher than the parent molecule in human cases of disseminated intravascular coagulation (DIC) initiated the following in vitro study. Following streptokinase induced plasma clot solubilization fibrinogen derivatives were investigated after ß-alanine precipitation of the plasma samples by polyacrylamide (PAA) gel electrophoresis, intra gel immunoprecipitation, two dimensional gel electrophoresis and by agarose gel filtration. Three fibrin-i degradation products were observed and characterized according to their relative electrophoretic mobility in 5% PAA gel: 0.23, 0.35, 0.46 (fibrinogen: 0.43) x 10-5 cm2/V x sec. They could also be demonstrated after electrophoresis in the presence of 5 M urea. Agarose gel filtration yielded one peak at 180 ml of effluent volume. The 0.23 derivative was eluted in the peak fractions, whilst the 0.35 and 0.46 derivatives were eluted together at approximately 201 ml of the effluent volume (fibrinogen: 225 ml). This indicates, that the three fibrin-i degradation products described are molecular entities with molecular weights higher than fibrinogen and, that the 0.46 derivative has an increased charge/molecular size ratio in comparison with fibrinogen. Corresponding data were obtained by two dimensional gel electrophoresis in gels of different pore size.

Ultrastructural Observations of the Rat Oocyte During the Estrous Cycle

1968 ◽  
Vol 26 ◽  
pp. 158-159
Author(s):  
I. M. Baccarini
Keyword(s):  
Electron Microscopy ◽  
Estrous Cycle ◽  
Uranyl Acetate ◽  
The Other ◽  
Osmic Acid ◽  
Acetate Solution ◽  
Lead Citrate ◽  
Vaginal Smears ◽  
Additional Information ◽  
Histochemical Methods

The cytological ultrastructure of the oocyte in different vertebrates and in insects has been described. Additional information on this subject is the purpose of this report which describes the findings on the oocyte of the rat during the estrous cycle.Vaginal smears were taken daily from 27 inbred Wistar-Furth rats to determine stages of the cycle. The animals were sacrificed with anesthesia in proestrous, estrous and metestrous and the ovaries removed surgically. In each case one ovary was used for electron microscopy and the other for histochemical methods.The ovary for electron microscopy was placed immediately into cold 3% phosphate-buffered glutaraldehyde for 2 hours and then into 1% phosphate-buffered osmic acid for 2-3 hours. The tissue was embedded in Durcupan ACM (FLUKA). The sections were double stained with 50% alcoholic uranyl acetate solution and with lead citrate.

Structural Investigation of Invertebrate Hemoglobins Using the Stem

1980 ◽  
Vol 38 ◽  
pp. 676-677
Author(s):  
O.H. Kapp ◽  
M. Ohtsuki ◽  
S.N. Vinogradov
Keyword(s):  
Scanning Transmission Electron Microscopy ◽  
Structural Investigation ◽  
Uranyl Acetate ◽  
Carbon Substrate ◽  
Distilled Water ◽  
Acetate Solution ◽  
Sedimentation Constant ◽  
Transmission Electron ◽  
Scanning Transmission ◽  
Polypeptide Chains

Scanning transmission electron microscopy (STEM) was used to study the structure of the extracellular hemoglobins of the annelids Lumbricus, Arenicola, and Nephtys. These are giant molecules, possessing a molecular weight of 3-4 x IO6,a sedimentation constant of about 60s and consisting of about five to seven different polypeptide chains (1,2,3). The samples were diluted to 100-200μg/ml with distilled water just before application to a very thin(∽15Å) carbon substrate supported on a microgrid. One percent (w/v) uranyl acetate solution was used for negative staining for 2 min. and dried in air. The specimens were examined with an electron energy of 22-35keV.

Intercellular Space of the Ovarian Follicles of the Rat During the Estrous Cycle

1967 ◽  
Vol 25 ◽  
pp. 60-61
Author(s):  
I. M. Baccarini ◽  
S. S. Glaczenski
Keyword(s):  
Electron Microscopy ◽  
Estrous Cycle ◽  
Intercellular Space ◽  
Amorphous Material ◽  
Single Layer ◽  
Primordial Follicle ◽  
Uranyl Acetate ◽  
Osmic Acid ◽  
Acetate Solution ◽  
Elastic Tissue

Vaginal smears were taken daily from inbred Wistar-Furth rats to determine the stage of the estrous cycle. A total of 27 animals were sacrificed by over-anesthesia with ether and the ovaries removed surgically. In each case one ovary was used for electron microscopy and the other for histochemical studies.The ovary for electron microscopy was placed immediately into cold 3% phosphate-buffered glutaraldehyde for 2 hours and then into 1% phosphate buffered osmic acid for 2-3 hours. The tissue was embedded in Durcupan ACM(Fluka). The sections were double stained with 50% alcoholic uranyl acetate solution and with lead citrate. The second ovary for histochemical studies was divided in half. One half was fixed in cold acetone for estimated alkaline phosphatase. The remaining tissue was fixed in 10% neutral formalin and stained with hematoxylin and eosin, PAS, Alcian blue, Gomori's trichrome, Gomori's aldehyde fuchsin for elastic tissue, and Wilder's reticulum. Follicles in various stages of development were studied. The intercellular space around the oocyte of the primordial follicle contains reticulum fibers. Bands of collagen fibers are seen between the oocyte and the single layer of cells (Figure 1). Between the basement membrane of the granulosa and theca cells in the secondary, tertiary, and graafian follicles, fibroblast-like cells with dilated cisterns are found. Amorphous material is present in the intercellular space (Figure 2).

The Use of Lead and Uranyl Ions As A Stain in Scanning Electron Microscopy

1976 ◽  
Vol 34 ◽  
pp. 314-315
Author(s):  
V. R. Mumaw ◽  
M. P. Goheen ◽  
B. L. Munger
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Uranyl Acetate ◽  
Distilled Water ◽  
Uranyl Ions ◽  
Cell Organelles ◽  
Lead Hydroxide ◽  
Definition Of ◽  
Cellular Components ◽  
Scanning Electron

Cellular components can be identified in cryofractured surfaces of osmium impregnated tissue examined in the scanning electron microscope (SEM). The present study compares the staining properties of uranyl and lead ions used to selectively enhance the definition of cell organelles. Tissues used for this study were liver, kidney and testis from normal rats. All of the tissue in this study was treated with osmium-thiocarbohydrazide-osmium (OTO) as described by Munger and Mumaw (1). Following the OTO treatment some tissues were placed in 1% aqueous uranyl acetate for 4 hrs. at room temp. Tissue stained with lead was placed in a Millonig's lead hydroxide solution for 2 hrs. Some tissue blocks were stained with uranyl acetate for 4 hrs. followed with 2 hrs. staining in lead hydroxide. Following each staining period the tissue was rinsed several times with distilled water before proceeding with the next step.

scholarly journals ULTRATHIN FROZEN SECTIONS

1967 ◽  
Vol 34 (3) ◽  
pp. 757-771 ◽  
Author(s):  
W. Bernhard ◽  
Elizabeth H. Leduc
Keyword(s):  
Electron Microscopy ◽  
Room Temperature ◽  
Osmium Tetroxide ◽  
Phosphotungstic Acid ◽  
Cultured Cells ◽  
Uranyl Acetate ◽  
Distilled Water ◽  
Frozen Sections ◽  
Simple Method ◽  
Ultrathin Frozen Sections

A relatively simple method for obtaining ultrathin, frozen sections for electron microscopy has been developed. Tissues, cultured cells, and bacteria may be employed. They are fixed in 1.25–4% glutaraldehyde for 1–4 hr, are washed overnight in buffer at 3°C, and are embedded in 20% thiolated gelatin or pure gelatin. Before sectioning they are partially dehydrated in 50% glycerol, frozen in liquid nitrogen on a modified tissue holder, and subsequently maintained at -70°C with dry ice. Finally, they are sectioned very rapidly with glass knives on a slightly modified Porter-Blum MT-1 microtome in a commercial deep-freeze maintained at -35°C and are floated in the trough of the knife on a 40% solution of dimethylsulfoxide (DMSO). The sections are picked up in plastic loops and transferred to distilled water at room temperature for thawing and removal of the DMSO, placed on grids coated with Formvar and carbon, air-dried, and stained with phosphotungstic acid, sodium silicotungstate, or a triple stain of osmium tetroxide, uranyl acetate, and lead. Large flat sections are obtained in which ultrastructural preservation is good. They are particularly useful for cytochemical studies.

Hexamethyldisilazane: A modified procedure

1989 ◽  
Vol 47 ◽  
pp. 1006-1007
Author(s):  
Verdon Laliberté ◽  
L.L. Hayes ◽  
B.M. Stanulis-Praeger
Keyword(s):  
Electron Microscopy ◽  
Room Temperature ◽  
Uranyl Acetate ◽  
Cell Contact ◽  
Critical Importance ◽  
Drying Procedures ◽  
Critical Point Drying ◽  
Scanning Electron ◽  
Better Than

Scanning electron microscopy is a powerful tool in the study of cell contact in vitro, and has shown, specifically, that fibroblasts under conditions of growth restriction increase cell contact by filopodia. Accurate quantitation of surface features like filopodia, however, depends particularly upon artifact-free drying procedures. Critical point drying (CPD) of fibroblast monolayers all too often causes cell flattening, shrinkage, loss of surface detail and cracking (Fig. 1). Newer methods utilizing hexamethyldisilazane (HMDS) and uranyl acetate (UA) preserve fibroblast contour and surface architecture better than CPD, but solvent choice in the preparation of UA is of critical importance. UA in 70% ethanol results in the formation of precipitate (Fig. 2) whether the specimens are in the cold (4°C) or at room temperature, overnight or for periods less than 1 hour regardless of filtering. It occurs when incubation is carried out in the dark and when it is followed by copious rinses in 70% ethanol.

scholarly journals Repeated use of uranyl acetate solution in section stainingin transmission electron microscopy

2005 ◽  
Vol 17 (1) ◽  
pp. 57-59 ◽  
Author(s):  
Masashi Yamaguchi ◽  
Masatoshi Shimizu ◽  
Tetsuro Yamaguchi ◽  
Misako Ohkusu ◽  
Susumu Kawamoto
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Uranyl Acetate ◽  
Acetate Solution ◽  
Transmission Electron ◽  
Repeated Use

Platinum Compounds as Stains for Electron Microscopy

1974 ◽  
Vol 32 ◽  
pp. 230-231
Author(s):  
S. K. Aggarwal ◽  
P. McAllister ◽  
R. W. Wagner ◽  
B. Rosenberg
Keyword(s):  
Electron Microscopy ◽  
Hela Cells ◽  
Uranyl Acetate ◽  
Sarcoma 180 ◽  
Platinum Compounds ◽  
Kb Cells ◽  
En Bloc ◽  
Human Lymphoma ◽  
Ultrathin Sections ◽  
Blue Coloration

Uranyl acetate has been used as an electron stain for en bloc staining as well as for staining ultrathin sections in conjunction with various lead stains (Fig. 1). Present studies reveal that various platinum compounds also show promise as electron stains. Certain platinum compounds have been shown to be effective anti-tumor agents. Of particular interest are the compounds with either uracil or thymine as one of the ligands (cis-Pt(II)-uracil; cis-Pt(II)-thymine). These compounds are amorphous, highly soluble in water and often exhibit an intense blue coloration. These compounds show enough electron density to be used as stains for electron microscopy. Most of the studies are based on various cell lines (human AV, cells, human lymphoma cells, KB cells, Sarcoma-180 ascites cells, chick fibroblasts and HeLa cells) while studies on tissue blocks are in progress.

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