title: Native immunogold labeling of cell surface proteins and viral glycoproteins for cryo-electron microscopy and cryo-electron tomography applications.
abstract: Numerous methods have been developed for immunogold labeling of thick, cryo-preserved biological specimens. However, most of the methods are permutations of chemical fixation and sample sectioning, which select and isolate the immunolabeled region of interest. We describe a method for combining immunogold labeling with cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET) of the surface proteins of intact mammalian cells or the surface glycoproteins of assembling and budding viruses in the context of virus-infected mammalian cells cultured on EM grids. In this method, the cells were maintained in culture media at physiologically relevant temperatures while sequentially incubated with the primary and secondary antibodies. Subsequently, the immunogold-labeled specimens were vitrified and observed under cryo-conditions in the transmission electron microscope. Cryo-EM and cryo-ET examination of the immunogold-labeled cells revealed the association of immunogold particles with the target antigens. Additionally, the cellular structure was unaltered by pre-immunolabeling chemical fixation and retained well-preserved plasma membranes, cytoskeletal elements, and macromolecular complexes. We think this technique will be of interest to cell biologists for cryo-EM and conventional studies of native cells and pathogen-infected cells.
authors.affiliation: Robert P. Apkarian Integrated Electron Microscopy Core, Emory University, Atlanta, Georgia (HY, ERW)
authors.forename: Hong
authors.initials: H
authors.lastname: Yi
authors.affiliation: Division of Pediatric Infectious Diseases, Emory University School of Medicine, Children's Healthcare of Atlanta, Atlanta, Georgia (JDS, RSD, CMH, KML, JEH, PWS, ERW)
authors.forename: Joshua D
authors.initials: JD
authors.lastname: Strauss
authors.affiliation: School of Biology, Georgia Institute of Technology, Atlanta, Georgia (ZK)
authors.forename: Zunlong
authors.initials: Z
authors.lastname: Ke
authors.affiliation: Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia (EA, PJS).
authors.forename: Eric
authors.initials: E
authors.lastname: Alonas
authors.affiliation: Division of Pediatric Infectious Diseases, Emory University School of Medicine, Children's Healthcare of Atlanta, Atlanta, Georgia (JDS, RSD, CMH, KML, JEH, PWS, ERW)
authors.forename: Rebecca S
authors.initials: RS
authors.lastname: Dillard
authors.affiliation: Division of Pediatric Infectious Diseases, Emory University School of Medicine, Children's Healthcare of Atlanta, Atlanta, Georgia (JDS, RSD, CMH, KML, JEH, PWS, ERW)
authors.forename: Cheri M
authors.initials: CM
authors.lastname: Hampton
authors.affiliation: Division of Pediatric Infectious Diseases, Emory University School of Medicine, Children's Healthcare of Atlanta, Atlanta, Georgia (JDS, RSD, CMH, KML, JEH, PWS, ERW)
authors.forename: Kristen M
authors.initials: KM
authors.lastname: Lamb
authors.affiliation: Division of Pediatric Infectious Diseases, Emory University School of Medicine, Children's Healthcare of Atlanta, Atlanta, Georgia (JDS, RSD, CMH, KML, JEH, PWS, ERW)
authors.forename: Jason E
authors.initials: JE
authors.lastname: Hammonds
authors.affiliation: Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia (EA, PJS).
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Santangelo
authors.affiliation: Division of Pediatric Infectious Diseases, Emory University School of Medicine, Children's Healthcare of Atlanta, Atlanta, Georgia (JDS, RSD, CMH, KML, JEH, PWS, ERW)
authors.forename: Paul W
authors.initials: PW
authors.lastname: Spearman
authors.affiliation: Robert P. Apkarian Integrated Electron Microscopy Core, Emory University, Atlanta, Georgia (HY, ERW)|Division of Pediatric Infectious Diseases, Emory University School of Medicine, Children's Healthcare of Atlanta, Atlanta, Georgia (JDS, RSD, CMH, KML, JEH, PWS, ERW)
authors.forename: Elizabeth R
authors.initials: ER
authors.lastname: Wright
mesh_terms: D000818:Animals; D002460:Cell Line; D020285:Cryoelectron Microscopy / Q000379:methods*; D055032:Electron Microscope Tomography / Q000379:methods*; D006023:Glycoproteins / Q000032:analysis*; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D008565:Membrane Proteins / Q000032:analysis*; D018357:Respiratory Syncytial Virus Infections / Q000821:virology; D012136:Respiratory Syncytial Viruses / Q000737:chemistry / Q000648:ultrastructure*; D014764:Viral Proteins / Q000032:analysis*
chemical_list: D006023:Glycoproteins; D008565:Membrane Proteins; D014764:Viral Proteins
keywords: cryo-electron microscopy (cryo-EM); cryo-electron tomography (cryo-ET); immuno-electron microscopy (immuno-EM); transmission electron microscopy (TEM)