title: Pre-embedding Double-Label Immunoelectron Microscopy of Chemically Fixed Tissue Culture Cells.
abstract: Localization of specific proteins within cells at the nanometer level of resolution is central to understanding how these proteins function in cell processes such as motility and intracellular trafficking. Such localization can be achieved by combining transmission electron microscopy (TEM) with immunogold labeling. Here we describe a pre-embedding, indirect gold immunolabeling approach to localize two different proteins of interest with secondary antibodies labeled with gold particles of different sizes in cells grown on cover slips. In this protocol, the cells are immunolabeled prior to being embedded in an epoxy resin for ultrathin sectioning. The protocol also includes strategies for optimizing the balance between ultrastructure and antigen preservation, steps to minimize nonspecific antibody binding, and steps to optimize antibody penetration.
authors: Department of Biological Sciences, The University of Memphis, 103 Life Sciences Bldg, Memphis, TN, 38152, USA.
authors: Lou G
authors: LG
authors: Boykins
authors: School of Molecular Biosciences, Washington State University, BLS 202F, 1770 NE Stadium Way, Pullman, WA, 99164, USA.
authors: Jonathan C R
authors: JC
authors: Jones
authors: Department of Biological Sciences, The University of Memphis, 103 Life Sciences Bldg, Memphis, TN, 38152, USA.
authors: Carlos E
authors: CE
authors: Estraño
authors: Department of Biological Sciences, The University of Memphis, 103 Life Sciences Bldg, Memphis, TN, 38152, USA.
authors: Steven D
authors: SD
authors: Schwartzbach
authors: Department of Biological Sciences, The University of Memphis, 103 Life Sciences Bldg, Memphis, TN, 38152, USA. oskalli@memphis.edu.
authors: Omar
authors: O
authors: Skalli
mesh_terms: D000906:Antibodies / Q000737:chemistry; D000941:Antigens / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D042783:Endothelial Cells / Q000378:metabolism / Q000648:ultrastructure*; D004853:Epoxy Resins / Q000737:chemistry; D015870:Gene Expression; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D039661:Integrin beta3 / Q000235:genetics* / Q000378:metabolism; D016253:Microscopy, Immunoelectron / Q000379:methods*; D008867:Microtomy; D013194:Staining and Labeling / Q000379:methods; D016610:Tissue Embedding / Q000379:methods*; D016707:Tissue Fixation / Q000379:methods; D014746:Vimentin / Q000235:genetics* / Q000378:metabolism
chemical_list: D000906:Antibodies; D000941:Antigens; D004853:Epoxy Resins; D039661:Integrin beta3; D014746:Vimentin
keywords: Antibodies; Immunogold; Monolayer; Pre-embedding