Analysis of Recombinant Adeno-Associated Virus (rAAV) Sample Morphology Using Negative Staining and High-Resolution Electron Microscopy
UMass Chan AffiliationsDepartment of Microbiology and Physiological Systems
Department of Neurology
Department of Radiology
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AbstractNegative staining is a simple and rapid method for studying the morphology and ultrastructure of small particulate specimens (e.g., viruses, bacteria, cell fragments, and isolated macromolecules such as proteins and nucleic acids). The technique described in this protocol involves allowing particles or fragments of cells to settle onto a support film, then applying a drop of metal salt solution to the adherent particulate specimen. The stain penetrates the interstices of the particles to bring out detail. In this situation, the preparation dries rapidly. The dissolved substance precipitates out of solution in an amorphous condition at the 0.1-nm level, and it is deposited over the support film and exposed surface of the specimen. The theoretical requirements of a good negative staining are a substance (1) of high density to provide high contrast, (2) at high solubility so that the stain does not come out of solution prematurely but does so only at the final stage of drying, (3) of high melting point and boiling point so that the material does not evaporate at high temperatures induced by the electron beam, and (4) in which the precipitate should be essentially amorphous down to the limit of resolution.
Hendricks G, Sena-Esteves M, Gao G. Analysis of Recombinant Adeno-Associated Virus (rAAV) Sample Morphology Using Negative Staining and High-Resolution Electron Microscopy. Cold Spring Harb Protoc. 2020 Aug 3;2020(8):095661. doi: 10.1101/pdb.prot095661. PMID: 32747581. Link to article on publisher's site