negative staining

Summary

Summary: The technique of washing tissue specimens with a concentrated solution of a heavy metal salt and letting it dry. The specimen will be covered with a very thin layer of the metal salt, being excluded in areas where an adsorbed macromolecule is present. The macromolecules allow electrons from the beam of an electron microscope to pass much more readily than the heavy metal; thus, a reversed or negative image of the molecule is created.

Top Publications

  1. Zhang L, Song J, Cavigiolio G, Ishida B, Zhang S, Kane J, et al. Morphology and structure of lipoproteins revealed by an optimized negative-staining protocol of electron microscopy. J Lipid Res. 2011;52:175-84 pubmed publisher
    ..Although electron microscopy (EM) is an alternative approach, conventional negative staining (NS) produces rouleau artifacts...
  2. Singh H, Donna Thompson B, Nickeleit V. Viral Haufen are urinary biomarkers of polyomavirus nephropathy: New diagnostic strategies utilizing negative staining electron microscopy. Ultrastruct Pathol. 2009;33:222-35 pubmed publisher
    ..Here, we describe technical details of Haufen detection by negative staining electron microscopy...
  3. Bremer A, Henn C, Engel A, Baumeister W, Aebi U. Has negative staining still a place in biomacromolecular electron microscopy?. Ultramicroscopy. 1992;46:85-111 pubmed
  4. Adrian M, Dubochet J, Fuller S, Harris J. Cryo-negative staining. Micron. 1998;29:145-60 pubmed
    ..of thin layers of vitrified biological suspensions in the presence of ammonium molybdate, which we term cryo-negative staining. The direct blotting of sample plus stain solution on holey carbon supports produces thin aqueous films ..
  5. Bangham A, Horne R. NEGATIVE STAINING OF PHOSPHOLIPIDS AND THEIR STRUCTURAL MODIFICATION BY SURFACE-ACTIVE AGENTS AS OBSERVED IN THE ELECTRON MICROSCOPE. J Mol Biol. 1964;8:660-8 pubmed
  6. El Bez C, Adrian M, Dubochet J, Cover T. High resolution structural analysis of Helicobacter pylori VacA toxin oligomers by cryo-negative staining electron microscopy. J Struct Biol. 2005;151:215-28 pubmed
    ..In the current study, vitrified VacA complexes were examined by cryo-negative staining electron microscopy, views of the different oligomeric structures in multiple orientations were classified ..
  7. De Carlo S, Boisset N, Hoenger A. High-resolution single-particle 3D analysis on GroEL prepared by cryo-negative staining. Micron. 2008;39:934-43 pubmed
    Cryo-negative staining was developed as a complementary technique to conventional cryo-electron microscopy on supramolecular complexes...
  8. Small J, Herzog M, Haner M, Abei U. Visualization of actin filaments in keratocyte lamellipodia: negative staining compared with freeze-drying. J Struct Biol. 1994;113:135-41 pubmed
    ..keratocyte, employing two procedures considered least damaging to actin filament arrays: freeze-drying and negative staining. At the front of the lamellipodium, where filaments density is the highest, freeze-dried images conveyed the ..
  9. Zhang L, Song J, Newhouse Y, Zhang S, Weisgraber K, Ren G. An optimized negative-staining protocol of electron microscopy for apoE4 POPC lipoprotein. J Lipid Res. 2010;51:1228-36 pubmed publisher
    ..The high contrast and fine detail of particle images produced using this optimized protocol lend themselves to the structural study of lipid-bound forms of apoE. ..

More Information

Publications62

  1. Laue M, Bannert N. Detection limit of negative staining electron microscopy for the diagnosis of bioterrorism-related micro-organisms. J Appl Microbiol. 2010;109:1159-68 pubmed publisher
    To determine the detection limit of diagnostic negative staining electron microscopy for the diagnosis of pathogens that could be used for bioterrorism...
  2. Woodcock C, Woodcock H, Horowitz R. Ultrastructure of chromatin. I. Negative staining of isolated fibers. J Cell Sci. 1991;99 ( Pt 1):99-106 pubmed
    ..Fibers allowed to unfold slightly in a buffer containing 50 mM monovalent ions show evidence of a two-stranded helix-like organization. These results are discussed in terms of current models for the structure of the chromatin fiber. ..
  3. De Carlo S, Harris J. Negative staining and cryo-negative staining of macromolecules and viruses for TEM. Micron. 2011;42:117-31 pubmed publisher
    In this review we cover the technical background to negative staining of biomolecules and viruses, and then expand upon the different possibilities and limitations...
  4. Pokutta S, Herrenknecht K, Kemler R, Engel J. Conformational changes of the recombinant extracellular domain of E-cadherin upon calcium binding. Eur J Biochem. 1994;223:1019-26 pubmed
    ..We suggest the calcium-dependent homoassociation therefore depends on additional effects connected with the cell surface association of E-cadherin. ..
  5. Signorell G, Chami M, Condemine G, Schenk A, Philippsen A, Engel A, et al. Projection maps of three members of the KdgM outer membrane protein family. J Struct Biol. 2007;160:395-403 pubmed
    ..The predicted transmembrane beta-barrels have a high similarity in the arrangement of the putative beta-strands and the loops, but do not match those of OmpG, a related protein porin whose structure was solved. ..
  6. Lambert O, Rigaud J. Phage DNA transfer into liposomes. Methods Enzymol. 2003;373:465-82 pubmed
  7. Ammar E, Fulton D, Bai X, Meulia T, Hogenhout S. An attachment tip and pili-like structures in insect- and plant-pathogenic spiroplasmas of the class Mollicutes. Arch Microbiol. 2004;181:97-105 pubmed
    ..Additionally, pili-like structures were observed by negative-staining TEM on the surface of Spiroplasma melliferum, and in thin sections of S. kunkelii infecting the leafhopper vector Dalbulus gelbus. ..
  8. Cabaleiro Lago C, Quinlan Pluck F, Lynch I, Dawson K, Linse S. Dual effect of amino modified polystyrene nanoparticles on amyloid ? protein fibrillation. ACS Chem Neurosci. 2010;1:279-87 pubmed publisher
    ..The overall rate of fibrillation will depend on the interplay between these two pathways, and the predominance of one mechanism over the other will be determined by the relative equilibrium and rate constants. ..
  9. Tan D, Hair Bejo M, Omar A, Aini I. Pathogenicity and molecular analysis of an infectious bursal disease virus isolated from Malaysian village chickens. Avian Dis. 2004;48:410-6 pubmed
    ..IBD infection in the village chicken may represent an important part of the IBD epidemiology because these birds could harbor the vvIBDV strain and should not be overlooked in the control and prevention of the disease. ..
  10. Monji A, Utsumi H, Yoshida I, Hashioka S, Tashiro K, Tashiro N. The relationship between Abeta-associated free radical generation and Abeta fibril formation revealed by negative stain electron microscopy and thioflavine-T fluorometric assay. Neurosci Lett. 2001;304:65-8 pubmed
    ..In conclusion, the present results suggest that Abeta-associated free radical generation is correlated with Abeta fibril formation while its generation is only observed transiently during the process of Abeta fibril formation. ..
  11. Miyagishima S, Takahara M, Kuroiwa T. Novel filaments 5 nm in diameter constitute the cytosolic ring of the plastid division apparatus. Plant Cell. 2001;13:707-21 pubmed
    ..Here, we present the ultrastructure of the outer ring at high resolution. To visualize the outer ring by negative staining, we isolated dividing chloroplasts from a synchronized culture of a red alga, Cyanidioschyzon merolae, and ..
  12. Desjardins P, Ban N, Mathews D, Kitasako J, Dodds J, McPherson A. Electron microscopy of satellite tobacco mosaic virus crystals: metal-coated, negatively stained and stereo pairs. J Electron Microsc (Tokyo). 2000;49:509-14 pubmed
    ..Conversely, knowing the exact unit cell parameters and the distribution of virions in the crystal from X-ray diffraction data aids in the further interpretation of electron micrographs of virus crystals...
  13. Kolodziej S, Penczek P, Stoops J. Utility of Butvar support film and methylamine tungstate stain in three-dimensional electron microscopy: agreement between stain and frozen-hydrated reconstructions. J Struct Biol. 1997;120:158-67 pubmed
  14. Morgan P, Hyman S, Rowe A, Mitchell T, Andrew P, Saibil H. Subunit organisation and symmetry of pore-forming, oligomeric pneumolysin. FEBS Lett. 1995;371:77-80 pubmed
    ..On the basis of these observations and our previous knowledge of the monomer domain structure we propose that the 4-domain subunits are packed in a square planar arrangement to form the pneumolysin oligomer. ..
  15. Fritz B, Moore K, Naides S. A combined pseudoreplica-immunochemical technique for research and diagnostic virology. Microsc Res Tech. 1992;21:59-64 pubmed
    ..Advantages include relative purification of samples, concentration of virus, morphological preservation, and enhanced diagnostic specificity. ..
  16. Braks I, Hoppert M, Roge S, Mayer F. Structural aspects and immunolocalization of the F420-reducing and non-F420-reducing hydrogenases from Methanobacterium thermoautotrophicum Marburg. J Bacteriol. 1994;176:7677-87 pubmed
    ..No linker particles were observed for this enzyme...
  17. Firtel M, Southam G, Harauz G, Beveridge T. Characterization of the cell wall of the sheathed methanogen Methanospirillum hungatei GP1 as an S layer. J Bacteriol. 1993;175:7550-60 pubmed
    ..The cell wall was composed of two nonglycosylated polypeptides (114 and 110 kDa). The cell wall resembled an archaeal S layer and may function in regulating the passage of small (< 10-kDa) sheath precursor proteins. ..
  18. Silverman J, Holter J, Berns L, Benning T, Neill J. Negative images due to clofazimine crystals simulating MAI infection in a bronchoalveolar lavage specimen. Diagn Cytopathol. 1993;9:534-9; discussion 539-40 pubmed
    ..We believe that this is the first report of clofazimine crystal deposition diagnosed in a BAL specimen along with electron microscopic examination of the cytologic material. ..
  19. Gaudin Y, Ruigrok R, Knossow M, Flamand A. Low-pH conformational changes of rabies virus glycoprotein and their role in membrane fusion. J Virol. 1993;67:1365-72 pubmed
    ..Our results suggest that this transient state is responsible for viral aggregation at low pH and could play a role in the first steps of the fusion mechanism. ..
  20. Lyon M, Marr K, Furcinitti P. Formation and characterization of two-dimensional crystals of photosystem II. J Struct Biol. 1993;110:133-40 pubmed
    ..The gap between the two PS II complexes was quite small, indicating that there may be a functional connection between the two halves of the dimer. ..
  21. Resch G, Urban E, Jacob S. The actin cytoskeleton in whole mount preparations and sections. Methods Cell Biol. 2010;96:529-64 pubmed publisher
    ..Techniques for three-dimensional visualisation of actin, protein localisation, and correlative light and electron microscopy are also included. ..
  22. Rachel R, Meyer C, Klingl A, G├╝rster S, Heimerl T, Wasserburger N, et al. Analysis of the ultrastructure of archaea by electron microscopy. Methods Cell Biol. 2010;96:47-69 pubmed publisher
    ..This is an important tool to make a link between biochemical and ultrastructural studies. ..
  23. Amano H, Hosaka Y. Morphological estimation of total number of influenza A type virion spikes. J Electron Microsc (Tokyo). 1992;41:104-6 pubmed
    When purified and stored influenza A type virions were observed by the negative staining method in electron microscopy, we found disintegrated virions associated with a population of dispersed but clustered spikes...
  24. Moores C. Studying microtubules by electron microscopy. Methods Cell Biol. 2008;88:299-317 pubmed publisher
    ..As more microtubule-associated proteins are uncovered, EM studies will make a significant contribution to our understanding of how these proteins influence microtubules and where in the cytoskeleton they act. ..
  25. Cheng A, Leung A, Fellmann D, Quispe J, Suloway C, Pulokas J, et al. Towards automated screening of two-dimensional crystals. J Struct Biol. 2007;160:324-31 pubmed
    ..While further development of these tools is certainly required for a turn-key system, the goal of fully automated screening appears to be within reach. ..
  26. Rao V, Leffers G. Purification and characterization of giant empty proheads from packaging-defective 23ptg mutants of bacteriophage T4. Virology. 1993;196:896-9 pubmed
    ..A purification protocol was developed to prepare large quantities of highly pure giant proheads. The purified giant proheads appeared to package both mature T4 DNA and concatemeric AD10 plasmid DNA in vitro. ..
  27. Messaoudi C, Boudier T, Lechaire J, Rigaud J, Delacroix H, Gaill F, et al. Use of cryo-negative staining in tomographic reconstruction of biological objects: application to T4 bacteriophage. Biol Cell. 2003;95:393-8 pubmed
    ..the present work we have tested, on T4 bacteriophage samples, the use of a new preparation technique, cryo-negative staining, which reduces the radiation damage while preserving a high signal-to-noise ratio...
  28. Furuno T, Sasabe H, Ikegami A. Imaging two-dimensional arrays of soluble proteins by atomic force microscopy in contact mode using a sharp supertip. Ultramicroscopy. 1998;70:125-31 pubmed
    ..The negative staining preparation conventionally used in the transmission electron microscopy of protein was applied and shown to ..
  29. Bergeron M, Boggavarapu R, Meury M, Ucurum Z, Caron L, Isenring P, et al. Frog oocytes to unveil the structure and supramolecular organization of human transport proteins. PLoS ONE. 2011;6:e21901 pubmed publisher
    ..This approach will open the way for the structure determination of many human membrane transporters taking full advantage of the Xenopus laevis oocyte expression system that generally yields robust functional expression. ..
  30. Benchimol M, Chang T, Alderete J. Trichomonas vaginalis: observation of coexistence of multiple viruses in the same isolate. FEMS Microbiol Lett. 2002;215:197-201 pubmed
    ..The sample was prepared by the negative staining carbon-film method directly onto freshly cleft mica. The detected viruses ranged in size from 33 to 200 nm...
  31. Kennaway C, Taylor J, Song C, Potrzebowski W, Nicholson W, White J, et al. Structure and operation of the DNA-translocating type I DNA restriction enzymes. Genes Dev. 2012;26:92-104 pubmed publisher
    ..The path followed by DNA through the complexes is revealed by using a DNA mimic anti-restriction protein. The structures reveal an evolutionary link between type I RM enzymes and type II RM enzymes. ..
  32. Ortolani F, Marchini M. Glutaraldehyde-induced D-band pattern of type II collagen fibrils as revealed by negative staining. Boll Soc Ital Biol Sper. 1993;69:107-13 pubmed
    A study was carried out on the negative staining band patterns (1% phosphotungstic acid (PTA) in phosphate buffer, 0.1 M, pH 7.4) exhibited by type II collagen fibrils after treatment with glutaraldehyde (5% in phosphate buffer 0...
  33. Kim L, Johnson M, Schmidt Krey I. Cryo-EM in the study of membrane transport proteins. Compr Physiol. 2012;2:283-93 pubmed publisher
    ..Further examples outline impressive results that were obtained by a combination of electron crystallography and X-ray crystallography as well as additional complementary methods. ..
  34. Sinclair K, Selinger L, D Silva C, Bae H, Yanke L, Cheng K. An improved method to illuminate and record a phytase assay in SDS-PAGE zymogram gels. J Biocommun. 2001;28:2-3 pubmed
    ..Increasing numbers of such PAGE gels are being submitted for photographic recording when it is believed that the image will be used for records, publication, scientific posters or AV presentations. ..
  35. Mabuchi K, Li Y, Carlos A, Wang C, Graceffa P. Caldesmon exhibits a clustered distribution along individual chicken gizzard native thin filaments. J Muscle Res Cell Motil. 2001;22:77-90 pubmed
    ..Similar clustering of CaD was also observed in plastic embedded tissue sections. These observations raise the possibility that CaD is not acting as a simple on/off switch, but more likely as a modulator, of smooth muscle contraction. ..
  36. Osawa T, Nozaka Y. Fine structure of the epidermal basement membrane of the lip: applications of dithiothreitol separation and ultrathin frozen sectioning. Acta Anat (Basel). 1995;153:106-10 pubmed
    ..Negatively stained ultrathin sections of dithiothreitol-separated specimens showed the fine structure of the lamina lucida. Bridging filaments in the lamina lucida were resolved by negative staining.
  37. Zhu Z, Cong W, Zhang P, Ma W, Min L, He H, et al. An expanding negative detection method for the visualization of DNA in polyacrylamide gels by eosin Y. Electrophoresis. 2010;31:3450-6 pubmed publisher
    ..In addition, the inclusion of inorganic salts in staining solution was also investigated, which has great effect on the staining efficiency. ..
  38. Milam S, Erickson H. Rapid in vitro assembly of Caulobacter crescentus FtsZ protein at pH 6.5 and 7.2. J Biol Chem. 2013;288:23675-9 pubmed publisher
    ..We conclude that CcFtsZ is not an outlier, but shows rapid assembly kinetics similar to FtsZ from related bacteria. ..
  39. Castellanos Serra L, Proenza W, Huerta V, Moritz R, Simpson R. Proteome analysis of polyacrylamide gel-separated proteins visualized by reversible negative staining using imidazole-zinc salts. Electrophoresis. 1999;20:732-7 pubmed
    ..This technique obviates the need for buffer exchange or gel lyophilisation due to all of the sample manipulation steps being carried out at near neutral pH and thus lends itself readily to automation. ..
  40. Arvanitakis L, Mesri E, Nador R, Said J, Asch A, Knowles D, et al. Establishment and characterization of a primary effusion (body cavity-based) lymphoma cell line (BC-3) harboring kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) in the absence of Epstein-Barr virus. Blood. 1996;88:2648-54 pubmed
  41. Namiki Y, Ueno K, Mitani H, Virtudazo E, Ohkusu M, Shimizu K, et al. Scanning and negative-staining electron microscopy of protoplast regeneration of a wild-type and two chitin synthase mutants in the pathogenic yeast Candida glabrata. J Electron Microsc (Tokyo). 2011;60:157-65 pubmed publisher
    ..These results suggest that CHS3A and CHS3B genes may have important roles in cell wall formation during protoplast regeneration, but can be compensated by other cell wall enzymes. ..
  42. Sousa A, Leapman R. Mass mapping of amyloid fibrils in the electron microscope using STEM imaging. Methods Mol Biol. 2013;950:195-207 pubmed publisher
    ..In this chapter we describe how the STEM technique can be applied to generate characteristic measurements of mass per length from isolated Alzheimer's amyloid fibrils. ..
  43. Bhardwaj A, Sankhala R, Olia A, Brooke D, Casjens S, Taylor D, et al. Structural Plasticity of the Protein Plug That Traps Newly Packaged Genomes in Podoviridae Virions. J Biol Chem. 2016;291:215-26 pubmed publisher
    ..The conformational plasticity of the tail needle N-terminal tip is built in the amino acid sequence, explaining its extraordinary conservation in nature. ..
  44. Nelson C, Minkkinen E, Bergkvist M, Hoelzer K, Fisher M, Bothner B, et al. Detecting small changes and additional peptides in the canine parvovirus capsid structure. J Virol. 2008;82:10397-407 pubmed publisher
    ..and evaluated their structural variation and permeability by protease sensitivity, spectrofluorometry, and negative staining electron microscopy...
  45. Kollman J, Quispe J. The 17A structure of the 420 kDa lobster clottable protein by single particle reconstruction from cryoelectron micrographs. J Struct Biol. 2005;151:306-14 pubmed
    ..The EM structure has been used in a low resolution molecular replacement search with data from orthorhombic CP crystals, and a solution is presented which describes the crystal packing. ..
  46. Newman M, Suk F, Cajimat M, Chua P, Shih C. Stability and morphology comparisons of self-assembled virus-like particles from wild-type and mutant human hepatitis B virus capsid proteins. J Virol. 2003;77:12950-60 pubmed
    ..Using negative staining and electron microscopy, full-length particles appear as "thick-walled" spherical particles with ..
  47. Kajava A, Cheng N, Cleaver R, Kessel M, Simon M, Willery E, et al. Beta-helix model for the filamentous haemagglutinin adhesin of Bordetella pertussis and related bacterial secretory proteins. Mol Microbiol. 2001;42:279-92 pubmed
    ..We have analysed a truncated variant of FHA by electron microscopy (negative staining, shadowing and scanning transmission electron microscopy of unstained specimens): these observations support ..
  48. Llorca O, Marco S, Carrascosa J, Valpuesta J. Symmetric GroEL-GroES complexes can contain substrate simultaneously in both GroEL rings. FEBS Lett. 1997;405:195-9 pubmed
    ..These results also suggest that symmetric GroEL-GroES complexes can use both rings simultaneously for folding, probably increasing the efficiency of the reaction. ..
  49. Makhov A, Boehmer P, Lehman I, Griffith J. Visualization of the unwinding of long DNA chains by the herpes simplex virus type 1 UL9 protein and ICP8. J Mol Biol. 1996;258:789-99 pubmed
    ..Here, electron microscopy has been applied to examine the mechanics of this reaction. Negative staining of the proteins revealed particles consisting primarily of ICP8 monomers and UL9 protein dimers...
  50. Ortolani F, Marchini M. Cartilage type II collagen fibrils show distinctive negative-staining band patterns differences between type II and type I unfixed or glutaraldehyde-fixed collagen fibrils. J Electron Microsc (Tokyo). 1995;44:365-75 pubmed
    ..b>Negative staining with phosphotungstic acid, pH 7...
  51. Heimburg T, Biltonen R. Thermotropic behavior of dimyristoylphosphatidylglycerol and its interaction with cytochrome c. Biochemistry. 1994;33:9477-88 pubmed
    ..Calorimetric titration studies of the binding of cytochrome c to DMPG indicate that protein binding is coupled cooperatively to changes in the state of the lipid.(ABSTRACT TRUNCATED AT 400 WORDS) ..
  52. Hauser N, Paulsson M. Native cartilage matrix protein (CMP). A compact trimer of subunits assembled via a coiled-coil alpha-helix. J Biol Chem. 1994;269:25747-53 pubmed
    ..The trimeric structure was retained after complete reduction under native conditions which shows that the coiled-coil domain is stable also in the absence of interchain disulfide bonds. ..
  53. Turner A, Wells B, Roberts K. Plasmodesmata of maize root tips: structure and composition. J Cell Sci. 1994;107 ( Pt 12):3351-61 pubmed
    ..A procedure is described for obtaining clean maize cell wall preparations that contain embedded plasmodesmata. Negative staining and rotary shadowing have been used with transmission electron microscopy to visualise the plasmodesmata in ..