A freeze substitution fixation-based gold enlarging technique for EM studies of endocytosed nanogold-labeled molecules
Abstract
We have developed methods to locate individual ligands that can be used for electron microscopy studies of dynamic events during endocytosis and subsequent intracellular trafficking. The methods are based on enlargement of 1.4 nm Nanogold attached to an endocytosed ligand. Nanogold, a small label that does not induce misdirection of ligand–receptor complexes, is ideal for labeling ligands endocytosed by live cells, but is too small to be routinely located in cells by electron microscopy. Traditional pre-embedding enhancement protocols to enlarge Nanogold are not compatible with high pressure freezing/freeze substitution fixation (HPF/FSF), the most accurate method to preserve ultrastructure and dynamic events during trafficking. We have developed an improved enhancement procedure for chemically fixed samples that reduced auto-nucleation, and a new pre-embedding gold enlarging technique for HPF/FSF samples that preserved contrast and ultrastructure and can be used for high-resolution tomography. We evaluated our methods using labeled Fc as a ligand for the neonatal Fc receptor. Attachment of Nanogold to Fc did not interfere with receptor binding or uptake, and gold-labeled Fc could be specifically enlarged to allow identification in 2D projections and in tomograms. These methods should be broadly applicable to many endocytosis and transcytosis studies.
Additional Information
© 2007 Elsevier Inc. Received 16 March 2007, Revised 5 July 2007, Accepted 17 July 2007, Available online 25 July 2007. We thank D.N. Mastronarde for advice on setting up SerialEM on the Caltech microscopes, E.T. O'Toole (University of Colorado) for help with IMOD, R.D. Powell (Nanoprobes, Inc.) for advice on gold enhancement, C.J. Ackerson and R.D. Kornberg (Stanford University) for preparing MPC gold bound to Fc for testing, and G.J. Jensen and W.F. Tivol (Caltech) for the use of the T12 microscope. This work was supported by the National Institutes of Health (2 R37 AI041239-06A1 to P.J.B.; RR000592 to J.R.M.) and gifts to Caltech to support electron microscopy from the Gordon and Betty Moore Foundation and the Agouron Institute. Author contributions: W.H. designed and implemented the HPF/FSF and chemical fixation gold enlarging methods, administered Au–Fc to rats, prepared tissue samples and performed the corresponding EM experiments. M.K.M. and J.R.M. developed the original concepts for FSF-based silver enhancement. C.K., S.M., A.M.G., D.B.T. and N.E.T. prepared Au–Fc conjugates. D.B.T. and N.E.T. did the confocal microscopy in FcRn-MDCK cells. M.K.M. and P.J.B. did the EM studies in MDCK cells at the Boulder Laboratory for 3D EM of Cells. P.J.B. supervised and planned the project. P.J.B., W.H. and J.R.M. jointly wrote the manuscript.Attached Files
Accepted Version - nihms-31728.pdf
Supplemental Material - mmc1__1_.doc
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Additional details
- PMCID
- PMC2076746
- Eprint ID
- 75435
- Resolver ID
- CaltechAUTHORS:20170327-143333776
- NIH
- 2 R37 AI041239-06A1
- NIH
- RR000592
- Gordon and Betty Moore Foundation
- Agouron Institute
- Created
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2017-03-28Created from EPrint's datestamp field
- Updated
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2021-11-15Created from EPrint's last_modified field