title: 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.
authors.affiliation: Division of Biology 114-96, California Institute of Technology, 1200 East California Boulevard, Pasadena, CA 91125, USA.
authors.forename: Wanzhong
authors.initials: W
authors.lastname: He
authors.forename: Christine
authors.initials: C
authors.lastname: Kivork
authors.forename: Suman
authors.initials: S
authors.lastname: Machinani
authors.forename: Mary K
authors.initials: MK
authors.lastname: Morphew
authors.forename: Anna M
authors.initials: AM
authors.lastname: Gail
authors.forename: Devin B
authors.initials: DB
authors.lastname: Tesar
authors.forename: Noreen E
authors.initials: NE
authors.lastname: Tiangco
authors.forename: J Richard
authors.initials: JR
authors.lastname: McIntosh
authors.forename: Pamela J
authors.initials: PJ
authors.lastname: Bjorkman
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D004705:Endocytosis; D005615:Freezing; D006046:Gold / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D053768:Metal Nanoparticles; D008854:Microscopy, Electron / Q000379:methods*; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D006046:Gold