Immunoelectron Microscopy of Cryofixed Freeze-Substituted Yeast

Fišerová, Jindřiška; Richardson, Christine; Goldberg, Martin W.

Abstract

Immunolabeling electron microscopy is a challenging technique with demands for perfect ultrastructural and antigen preservation. High-pressure freezing offers an excellent way to fix cellular structure. However, its use for immunolabeling has remained limited because of the low frequency of labeling due to loss of protein antigenicity or accessibility. Here we present a protocol for immunogold labeling of the yeast Saccharomyces cerevisiae that gives specific and multiple labeling while keeping the finest structural details. We use the protocol to reveal the organization of individual nuclear pore complex proteins and the position of transport factors in the yeast Saccharomyces cerevisiae in relation to actual transport events.

Citation

Fišerová, J., Richardson, C., & Goldberg, M. W. (2016). Immunoelectron Microscopy of Cryofixed Freeze-Substituted Yeast. In S. D. Schwartzbach, O. Skalli, & T. Schikorski (Eds.), High-Resolution Imaging of Cellular Proteins (243-258). Humana Press. https://doi.org/10.1007/978-1-4939-6352-2_15

• Online Publication Date: Aug 12, 2016
• Publication Date: 2016-08
• Deposit Date: Oct 13, 2016
• Publisher: Humana Press
• Pages: 243-258
• Series Title: Methods in Molecular Biology
• Series Number: 1474
• Book Title: High-Resolution Imaging of Cellular Proteins
• ISBN: 9781493963508
• DOI: https://doi.org/10.1007/978-1-4939-6352-2_15