title: Immunolocalization of Proteins in Fission Yeast by Electron Microscopy.
abstract: Electron microscopy (EM) immunolocalization of antigens in fission yeast can be accomplished with cells processed by rapid freezing and freeze-substitution followed by embedding in acrylic or methacrylate resins. Microtome sections of embedded cells are collected onto EM grids. Primary antibodies to the antigen of interest, followed by secondary antibodies conjugated to colloidal gold, are allowed to bind to antigens at the surface of these plastic sections. This type of postembed labeling provides information on antigen localization to a resolution of 10-20 nm, depending on the size of the metal particle used, the form of the antibody (Fab vs. complete IgG or IgM), and whether direct or indirect labeling is used. The method has the potential to map macromolecules in three dimensions in a relatively large volume when thin (30-60-nm) serial sections are labeled, imaged, aligned, and modeled to create a representative volume. The biggest challenge of this technique is the necessary compromise between the preservation of cellular ultrastructure and the preservation of antigen reactivity. The protocols described here show how to immunolabel samples for EM and include suggestions for overcoming challenges related to antigen preservation.
authors.affiliation: Laboratory for 3D Electron Microscopy, University of Colorado, Boulder, Colorado 80309-0347.
authors.forename: Mary K
authors.initials: MK
authors.lastname: Morphew
authors.affiliation: Laboratory for 3D Electron Microscopy, University of Colorado, Boulder, Colorado 80309-0347.
authors.forename: Thomas H
authors.initials: TH
authors.lastname: Giddings
authors.affiliation: Laboratory for 3D Electron Microscopy, University of Colorado, Boulder, Colorado 80309-0347.|Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado 80309-0347.
authors.forename: J Richard
authors.initials: JR
authors.lastname: McIntosh
mesh_terms: D000908:Antibodies, Fungal / Q000378:metabolism; D005615:Freezing; D005656:Fungal Proteins / Q000032:analysis*; D007150:Immunohistochemistry / Q000379:methods*; D016253:Microscopy, Immunoelectron / Q000379:methods*; D015388:Organelles / Q000737:chemistry*; D016613:Plastic Embedding; D012568:Schizosaccharomyces / Q000737:chemistry* / Q000648:ultrastructure*
chemical_list: D000908:Antibodies, Fungal; D005656:Fungal Proteins