Morphew, M. and He, W. and Bjorkman, P. J. and McIntosh, J. Richard (2008) Silver enhancement of Nanogold particles during freeze substitution for electron microscopy. Journal of Microscopy, 230 (2). pp. 263-267. ISSN 0022-2720. http://resolver.caltech.edu/CaltechAUTHORS:20100721-114957210
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Recent advances in rapid freezing and fixation by freeze substitution have allowed structural cell biologists to apply these reliable modes of sample preparation to a wide range of specimens and scientific problems. Progress in electron tomography has produced cellular images with resolution approaching 4 nm in 3D, but our ability to localize macromolecules in these well-fixed, well-resolved samples has remained limited. When light fixation and low temperature embedding are employed with appropriate resins, immuno-localizations can recognize antigens at a section's surface, but labelling is therefore confined, not throughout the section's depth. Small, electron-dense markers, like Nanogold®, will often enter a living cell, serving as reliable tracers for endocytic activity, but these markers are usually too small to be visible in the context of a cell. We have developed a method for the silver enhancement of Nanogold particles that works during freeze substitution in organic solvents at low temperature. Here, we describe the development of this method, based on in vitro tests of reagents and conditions. We then show results from application of the method to an in vivo system, using Nanogold to track the internalization of immunoglobulin by neonatal murine intestinal epithelium, a specific example of receptor-mediated membrane traffic.
|Additional Information:||© 2008 Royal Microscopical Society. Received 12 April 2007; accepted 13 July 2007. We gratefully acknowledge Eileen O’Toole for assistance with modelling. This work was supported in part by RR00592 from the NIH to JRM and 2R37 AI041239-06A1 to P.J.B.|
|Subject Keywords:||Cytochemistry; endocytosis; immunoEM; immunogold|
|Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Jason Perez|
|Deposited On:||30 Jul 2010 18:11|
|Last Modified:||26 Dec 2012 12:15|
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