A freeze substitution fixation-based gold enlarging technique for EM studies of endocytosed Nanogold-labeled molecules

• Published in: Journal of structural biology Vol. 160; no. 1; pp. 103 - 113
• Main Authors: He, Wanzhong, Kivork, Christine, Machinani, Suman, Morphew, Mary K., Gail, Anna M., Tesar, Devin B., Tiangco, Noreen E., McIntosh, J. Richard, Bjorkman, Pamela J.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01-10-2007
• Subjects: Animals; Animals, Newborn; Electron microscopy; Electron tomography; Endocytosis; Fc receptor; Freeze substitution fixation; Freezing; Gold - chemistry; Gold enhancement; High pressure freezing; Hydrogen-Ion Concentration; IgG; Metal Nanoparticles; Microscopy, Electron - methods; Nanogold; Rats; Rats, Sprague-Dawley; Transcytosis; Endocytosis; Fc receptor; IgG; Electron tomography; Transcytosis; Electron microscopy; Nanogold; Freeze substitution fixation; Gold enhancement; High pressure freezing
• ISSN: 1047-8477; 1095-8657
• DOI: 10.1016/j.jsb.2007.07.004

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 1.4nm 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.