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J. Robin Harris - One of the best experts on this subject based on the ideXlab platform.
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Negative Staining and cryo-Negative Staining: applications in biology and medicine.
Methods of Molecular Biology, 2013Co-Authors: J. Robin Harris, Sacha De CarloAbstract:Negative Staining is widely applicable to isolated viruses, protein molecules, macromolecular assemblies and fibrils, subcellular membrane fractions, liposomes and artificial membranes, synthetic DNA arrays, and also to polymer solutions and a variety of nanotechnology samples. Techniques are provided for the preparation of the necessary support films (continuous carbon and holey/perforated carbon). The range of suitable Negative stains is presented, with some emphasis on the benefit of using ammonium molybdate and of Negative stain-trehalose combinations. Protocols are provided for the single droplet Negative Staining technique (on continuous and holey carbon support films), the floating and carbon sandwich techniques in addition to the Negative Staining-carbon film (NS-CF) technique for randomly dispersed fragile molecules, 2D crystallization of proteins and for cleavage of cells and organelles. Immuno-Negative Staining and Negative Staining of affinity labeled complexes (e.g., biotin-streptavidin) are presented in some detail. The formation of immune complexes in solution for droplet Negative Staining is given, as is the use of carbon-plastic support films as an adsorption surface on which to perform immunolabeling or affinity experiments, prior to Negative Staining. Dynamic biological systems can be investigated by Negative Staining, where the time period is in excess of a few minutes, but there are possibilities to greatly reduce the time by rapid stabilization of molecular systems with uranyl acetate or tannic acid. The more recently developed cryo-Negative Staining procedures are also included: first, the high concentration ammonium molybdate procedure on holey carbon films and second, the carbon sandwich procedure using uranyl formate. Several electron micrographs showing examples of applications of Negative Staining techniques are included and the chapter is thoroughly referenced.
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Negative Staining and cryo-Negative Staining of macromolecules and viruses for TEM.
Micron, 2010Co-Authors: Sacha De Carlo, J. Robin HarrisAbstract:In this review we cover the technical background to Negative Staining of biomolecules and viruses, and then expand upon the different possibilities and limitations. Topics range from conventional air-dry Negative Staining of samples adsorbed to carbon support films, the variant termed the "Negative Staining-carbon film" technique and Negative Staining of samples spread across the holes of holey-carbon support films, to a consideration of dynamic/time-dependent Negative Staining. For each of these approaches examples of attainable data are given. The cryo-Negative Staining technique for the specimen preparation of frozen-hydrated/vitrified samples is also presented. A detailed protocol to successfully achieve cryo-Negative Staining with ammonium molybdate is given, as well as examples of data, which support the claim that cryo-Negative Staining provides a useful approach for the high-resolution study of macromolecular and viral structure.
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Negative Staining across holes: application to fibril and tubular structures.
Micron, 2007Co-Authors: J. Robin HarrisAbstract:The Negative Staining technique, when used with holey carbon support films, presents superior imaging conditions than is the case when samples are adsorbed to continuous carbon films. A demonstration of this Negative Staining approach is presented, using ammonium molybdate in combination with trehalose, applied to several fibrillar and tubular samples. Fibrils formed from the amyloid-β peptide and the protease inhibitor pepstain A spread very well unsupported across holes and the different polymorphic fibril forms can be readily assessed. However, tubular forms of amyloid-β have a tendency to be flattened, due to surface tension forces prior to and during specimen drying. Sub-fibril assembly forms and D-banded rat tail type 1 collagen fibres are presented. The air-dried collagen images produced are shown to contain almost as much detail as those obtainable by cryo-Negative Staining. Fragile DNA and DNA-protein nanotubes are also shown to yield superior quality images to those produced on continuous carbon films. The iron-storage protein, frataxin, creates elongated oligomeric assemblies, containing bound ferrihydrite microcrystals. The iron particles within these flexuous oligomers can be defined in the presence of ammonium molybdate, but they are more readily demonstrated if the frataxin is spread across holes in the presence of trehalose alone. The samples used here serve to show the likely benefit obtainable from Negative Staining across holes for a range of other fibrillar and tubular samples in biology, medicine and nanobiotechnology.
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Negative Staining of thinly spread biological samples.
Methods in molecular biology (Clifton N.J.), 2007Co-Authors: J. Robin HarrisAbstract:Negative Staining is widely applicable to isolated viruses, protein molecules, macro-molecular assemblies and fibrils, subcellular membrane fractions, liposomes and artificial membranes, synthetic DNA arrays, and also to polymer solutions. In this chapter, techniques are provided for the preparation of the necessary support films (continuous carbon and holey/perforated carbon). The range of suitable Negative stains is presented, with some emphasis on the benefit of using ammonium molybdate and of Negative stain-trehalose combinations. Protocols are provided for the single-droplet Negative Staining technique (on continuous and holey carbon support films), the Negative Staining-carbon film technique, for randomly dispersed fragile molecules, 2D crystallization of proteins, and for cleavage of cells and organelles. The newly developed cryoNegative Staining procedure also is included. ImmunoNegative Staining and Negative Staining of affinity labeled complexes (e.g., biotin-streptavidin) are discussed in some detail. The formation of immune complexes in solution for droplet Negative Staining is presented, as is the use of carbon-plastic support films as an adsorption surface on which to perform immunolabeling or affinity experiments, before Negative Staining. Dynamic biological systems can be investigated by Negative Staining, where the time period is in excess of a few minutes, but there are possibilities to greatly reduce the time by rapid stabilization of molecular systems with uranyl acetate or tannic acid.
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Cryo-Negative Staining
Micron, 1998Co-Authors: Marc Adrian, Jacques Dubochet, Stephen D. Fuller, J. Robin HarrisAbstract:Abstract A procedure is presented for the preparation of thin layers of vitrified biological suspensions in the presence of ammonium molybdate, which we termcryo-Negative Staining. The direct blotting of sample plus stain solution on holey carbon supports produces thin aqueous films across the holes, which are routinely thiner than the aqueous film produced by conventional Negative Staining on a continuous carbon layer. Because of this, a higher than usual concentration of Negative stain (ca. 16% rather than 2%) is required for cryo-Negative Staining in order to produce an optimal image contrast. The maintenance of the hydrated state, the absence of adsorption to a carbon film and associated sample flattening, together with reduced stain granularity, generates high contrast cryo-images of superior quality to conventional air-dry Negative Staining. Image features characteristic of unstained vitrified cryo-electron microscopic specimens are present, but with reverse contrast. Examples of cryo-Negative Staining of several particulate biological samples are shown, including bacteriophage T2, tobacco mosaic virus (TMV), bovine liver catalase crystals, tomato bushy stunt virus (TBSV), turnip yellow mosaic virus (TYMV), keyhole limpet hemocyanin (KLH) types 1 and 2, the 20S proteasome from moss and theE. coli chaperone GroEL. Densitometric quantitation of the mass-density of cryo-Negatively stained bacteriophage T2 specimens before and after freeze-drying within the TEM indicates a water content of 30% in the vitreous specimen. Determination of the image resolution from cryo-Negatively stained TMV rods and catalase crystals shows the presence of optical diffraction data to ca. 10A˚and 115A˚, respectively. For cryo-Negatively stained vitrified catalase crystals, electron diffraction shows that atomic resolution is preserved (to better than 20 diffraction orders and less than 3A˚). The electron diffraction resolution is reduced to ca. 10A˚when catalase crystal specimens are prepared without freezing or when they are freeze-dried in the electron microscope. Thin vitrified films of TMV, TBSV and TYMV in the presence of 16% ammonium molybdate show a clear indication of two-dimensional (2-D) order, confirmed by single particle orientational analysis of TBSV and 2-D crystallographic analysis of TYMV. These observations are in accord with earlier claims that ammonium molybdate induces 2-D array and crystal formation from viruses and macromolecules during drying onto mica. Three-dimensional analysis of the TBSV sample using the tools of icosahedral reconstruction revealed that a significant fraction of the particles were distorted. A reconstruction from a subset of undistorted particles produced the characteristic T=3 dimer clustered structure of TBSV, although the spikes are shortened relative to the structure defined by X-ray crystallography. The 20S proteasome, GroEL, catalase, bacteriophage T2, TMV, TBSV and TYMV all show no indication of sample instability during cryo-Negative Staining. However, detectable dissociation of the KLH2 oligomers in the presence of the high concentration of ammonium molybdate conforms with existing knowledge on the molybdate-induced dissociation of this molecule. This indicates that the possibility of sample-stain interaction in solution, prior to vitrification, must always be carefully assessed.
Sacha De Carlo - One of the best experts on this subject based on the ideXlab platform.
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Negative Staining and cryo-Negative Staining: applications in biology and medicine.
Methods of Molecular Biology, 2013Co-Authors: J. Robin Harris, Sacha De CarloAbstract:Negative Staining is widely applicable to isolated viruses, protein molecules, macromolecular assemblies and fibrils, subcellular membrane fractions, liposomes and artificial membranes, synthetic DNA arrays, and also to polymer solutions and a variety of nanotechnology samples. Techniques are provided for the preparation of the necessary support films (continuous carbon and holey/perforated carbon). The range of suitable Negative stains is presented, with some emphasis on the benefit of using ammonium molybdate and of Negative stain-trehalose combinations. Protocols are provided for the single droplet Negative Staining technique (on continuous and holey carbon support films), the floating and carbon sandwich techniques in addition to the Negative Staining-carbon film (NS-CF) technique for randomly dispersed fragile molecules, 2D crystallization of proteins and for cleavage of cells and organelles. Immuno-Negative Staining and Negative Staining of affinity labeled complexes (e.g., biotin-streptavidin) are presented in some detail. The formation of immune complexes in solution for droplet Negative Staining is given, as is the use of carbon-plastic support films as an adsorption surface on which to perform immunolabeling or affinity experiments, prior to Negative Staining. Dynamic biological systems can be investigated by Negative Staining, where the time period is in excess of a few minutes, but there are possibilities to greatly reduce the time by rapid stabilization of molecular systems with uranyl acetate or tannic acid. The more recently developed cryo-Negative Staining procedures are also included: first, the high concentration ammonium molybdate procedure on holey carbon films and second, the carbon sandwich procedure using uranyl formate. Several electron micrographs showing examples of applications of Negative Staining techniques are included and the chapter is thoroughly referenced.
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Negative Staining and cryo-Negative Staining of macromolecules and viruses for TEM.
Micron, 2010Co-Authors: Sacha De Carlo, J. Robin HarrisAbstract:In this review we cover the technical background to Negative Staining of biomolecules and viruses, and then expand upon the different possibilities and limitations. Topics range from conventional air-dry Negative Staining of samples adsorbed to carbon support films, the variant termed the "Negative Staining-carbon film" technique and Negative Staining of samples spread across the holes of holey-carbon support films, to a consideration of dynamic/time-dependent Negative Staining. For each of these approaches examples of attainable data are given. The cryo-Negative Staining technique for the specimen preparation of frozen-hydrated/vitrified samples is also presented. A detailed protocol to successfully achieve cryo-Negative Staining with ammonium molybdate is given, as well as examples of data, which support the claim that cryo-Negative Staining provides a useful approach for the high-resolution study of macromolecular and viral structure.
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High-resolution single-particle 3D analysis on GroEL prepared by cryo-Negative Staining.
Micron, 2007Co-Authors: Sacha De Carlo, N. Boisset, Andreas HoengerAbstract:Cryo-Negative Staining was developed as a complementary technique to conventional cryo-electron microscopy on supramolecular complexes. It allows imaging biological samples in a comparable state of structural preservation to conventional cryo-EM but the Staining produces better contrast in accessible areas and allows data recording at lower defocus values. Cryo-Negative Staining vitrifies biological particles in the presence of a concentrated ammonium molybdate solution at neutral pH. It was successfully used to study the structure and dynamics of several macromolecules, such as human transcription factors and RNA polymerases. Imaging macromolecular complexes with cryo-Negative Staining has been established previously to better than 2 nm detail. However, it has not been verified so far whether cryo-Negative Staining also visualizes secondary structure elements. Using the well known E. coli GroEL chaperonin, we could show that the structure is well preserved to ∼10 A resolution. Secondary structure details are at least partially resolved in the electron density map.
William H. Massover - One of the best experts on this subject based on the ideXlab platform.
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On the experimental use of light metal salts for Negative Staining.
Microscopy and Microanalysis, 2008Co-Authors: William H. MassoverAbstract:All common Negative stains are salts of heavy metals. To remedy several technical defects inherent in the use of heavy metal compounds, this study investigates whether salts of the light metals sodium, magnesium, and aluminum can function as Negative stains. Screening criteria require aqueous solubility at pH 7.0, formation of a smooth amorphous layer upon drying, and transmission electron microscope imaging of the 87-A (8.7-nm) lattice periodicity in thin catalase crystals. Six of 23 salts evaluated pass all three screens; detection of the protein shell in ferritin macromolecules indicates that light metal salts also provide Negative Staining of single particle specimens. Appositional contrast is less than that given by heavy metal Negative stains; image density can be raised by increasing electron phase contrast and by selecting salts with phosphate or sulfate anions, thereby adding strong scattering from P or S atoms. Low-dose electron diffraction of catalase crystals Negatively stained with 200 mM magnesium sulfate shows Bragg spots extending out to 4.4 A. Future experimental use of sodium phosphate buffer and magnesium sulfate for Negative Staining is anticipated, particularly in designing new cocktail (multicomponent) Negative stains able to support and protect protein structure to higher resolution levels than are currently achieved.
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Negative Staining permits 4.0 A resolution with low-dose electron diffraction of catalase crystals.
Ultramicroscopy, 2001Co-Authors: William H. Massover, Philip MarshAbstract:Abstract Low-dose electron diffraction of thin single crystals of catalase that are Negatively stained with the light-atom compound, dipotassium glucose-1,6-diphosphate, reveals Bragg reflections extending to 4.0 A (=0.40 nm). Under the same conditions, Negative Staining with the traditional heavy-metal salt, ammonium molybdate, also gives diffraction spots extending to 4.0 A. These results establish that Negative Staining of protein crystals preserves periodic structural information into the high-resolution range, unlike the widely accepted current belief that this methodology can give a resolution limited to only 20–25 A.
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Is Negative Staining Capable of High Resolution
Microscopy and Microanalysis, 2001Co-Authors: William H. MassoverAbstract:Everyone presently answers “no” to this question, since this methodology for specimen preparation and preservation commonly is believed to be inherently limited to 20-25Å (2.0-2.5nm) resolution. Achievement of resolution levels smaller than this figure have been published only rarely. This report presents experimental evidence from electron diffraction showing that Negative Staining can preserve periodic protein structure to the level of at least 4Å.A suspension of bovine liver catalase crystals (orthorhombic: a= 69Å, b= 174Å, c= 206 Å [1]) is deposited on a hydrophilic carbon support and Negatively stained using an on-grid protocol [2,3]. For low-dose electron diffraction with a JEOL 100CX transmission electron microscope (l00kV), the set-up for routine selected area diffraction is modified to keep the second condenser lens maximally overfocused; by a defocusing of the diffraction pattern, the grid can be surveyed and suitable thin single crystals centered in the diffraction aperture with only negligible irradiation.
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Unconventional Negative stains: Heavy metals are not required for Negative Staining
Ultramicroscopy, 1997Co-Authors: William H. Massover, Philip MarshAbstract:Abstract Salts of heavy metals (tungsten, uranium, and molybdenum) have long been used as Negative stains for the transmission electron microscopy of protein molecules, supramolecular assemblies, and viruses, Negative Staining still reveals details about protein structure only to around 25 A (= 2.5 nm), most likely due to several major technical deficiencies in all traditional stain reagents. Experimental tests of unconventional compounds show that potassium aluminum sulfate, ammonium borate, and sodium tetraborate all dry into a glassy layer, and provide sufficient contrast to image the 86 A lattice periods in thin crystals of catalase. Computer-generated power spectra of high-dose images indicate that sodium tetraborate can preserve periodic order in crystalline catalase to the same level (i.e., 28 A) as the heavy metal Negative stain, sodium silicotungstate. Sodium tetraborate also reveals the 40 A wide central channel within tobacco mosaic virus, and the 25 A thick protein shell of ferritin molecules. Images with these light atom compounds should be formed with a decreased electron beam exposure, in order to preclude the radiation-induced formation of voids within the dried stain. The results establish that heavy metal constituents are not necessary for a compound to function as a Negative stain, and suggest that it should be possible to identify new reagents having the missing desired properties needed to obtain higher resolution imaging of protein structure with Negative Staining.
Michele Carbone - One of the best experts on this subject based on the ideXlab platform.
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A cytokeratin- and calretinin-Negative Staining sarcomatoid malignant mesothelioma.
Anticancer Research, 2004Co-Authors: Michael G. Hurtuk, Michele CarboneAbstract:Malignant Mesothelioma, or mesothelioma, is a mesothelial-based malignancy that may occur in the pleura, pericardium and peritoneum. Mesothelioma is a very aggressive cancer with limited treatment, and a median survival of about 1 year. At times, the diagnosis of mesothelioma may be problematic. The final diagnosis of mesothelioma relies on histology and often is dependent upon immunohistochemistry. It is generally assumed that mesotheliomas must stain positive for cytokeratin and calretinin and Negative Staining for these markers would rule out the diagnosis. We encountered a patient with a pleural-based, cytokeratin- and calretinin-Negative sarcomatoid malignancy. These Negative Stainings would rule out the diagnosis of mesothelioma but, after careful consideration of the patient's clinical records, and additional histological and immunohistochemical studies, we conclude that this patient suffered from mesothelioma of the sarcomatoid type.
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A cytokeratin- and calretinin-Negative Staining sarcomatoid malignant mesothelioma.
Anticancer Research, 2004Co-Authors: Michael G. Hurtuk, Michele CarboneAbstract:Malignant Mesothelioma, or mesothelioma, is a mesothelial-based malignancy that may occur in the pleura, pericardium and peritoneum. Mesothelioma is a very aggressive cancer with limited treatment, and a median survival of about 1 year. At times, the diagnosis of mesothelioma may be problematic. The final diagnosis of mesothelioma relies on histology and often is dependent upon immunohistochemistry. It is generally assumed that mesotheliomas must stain positive for cytokeratin and calretinin and Negative Staining for these markers would rule out the diagnosis. We encountered a patient with a pleural-based, cytokeratin- and calretinin- Negative sarcomatoid malignancy. These Negative Stainings would rule out the diagnosis of mesothelioma but, after careful consideration of the patient's clinical records, and additional histological and immunohistochemical studies, we conclude that this patient suffered from mesothelioma of the sarcomatoid type. Mesothelial cells form the lining of the pericardial, pleural and peritoneal cavities. They are among the most
Philip Marsh - One of the best experts on this subject based on the ideXlab platform.
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Negative Staining permits 4.0 A resolution with low-dose electron diffraction of catalase crystals.
Ultramicroscopy, 2001Co-Authors: William H. Massover, Philip MarshAbstract:Abstract Low-dose electron diffraction of thin single crystals of catalase that are Negatively stained with the light-atom compound, dipotassium glucose-1,6-diphosphate, reveals Bragg reflections extending to 4.0 A (=0.40 nm). Under the same conditions, Negative Staining with the traditional heavy-metal salt, ammonium molybdate, also gives diffraction spots extending to 4.0 A. These results establish that Negative Staining of protein crystals preserves periodic structural information into the high-resolution range, unlike the widely accepted current belief that this methodology can give a resolution limited to only 20–25 A.
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Unconventional Negative stains: Heavy metals are not required for Negative Staining
Ultramicroscopy, 1997Co-Authors: William H. Massover, Philip MarshAbstract:Abstract Salts of heavy metals (tungsten, uranium, and molybdenum) have long been used as Negative stains for the transmission electron microscopy of protein molecules, supramolecular assemblies, and viruses, Negative Staining still reveals details about protein structure only to around 25 A (= 2.5 nm), most likely due to several major technical deficiencies in all traditional stain reagents. Experimental tests of unconventional compounds show that potassium aluminum sulfate, ammonium borate, and sodium tetraborate all dry into a glassy layer, and provide sufficient contrast to image the 86 A lattice periods in thin crystals of catalase. Computer-generated power spectra of high-dose images indicate that sodium tetraborate can preserve periodic order in crystalline catalase to the same level (i.e., 28 A) as the heavy metal Negative stain, sodium silicotungstate. Sodium tetraborate also reveals the 40 A wide central channel within tobacco mosaic virus, and the 25 A thick protein shell of ferritin molecules. Images with these light atom compounds should be formed with a decreased electron beam exposure, in order to preclude the radiation-induced formation of voids within the dried stain. The results establish that heavy metal constituents are not necessary for a compound to function as a Negative stain, and suggest that it should be possible to identify new reagents having the missing desired properties needed to obtain higher resolution imaging of protein structure with Negative Staining.
