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Pre-embedding Double-Label Immunoelectron Microscopy of Chemically Fixed Tissue Culture Cells.
Boykins, Lou G; Jones, Jonathan C R; Estraño, Carlos E; Schwartzbach, Steven D; Skalli, Omar.
Afiliação
  • Boykins LG; Department of Biological Sciences, The University of Memphis, 103 Life Sciences Bldg, Memphis, TN, 38152, USA.
  • Jones JC; School of Molecular Biosciences, Washington State University, BLS 202F, 1770 NE Stadium Way, Pullman, WA, 99164, USA.
  • Estraño CE; Department of Biological Sciences, The University of Memphis, 103 Life Sciences Bldg, Memphis, TN, 38152, USA.
  • Schwartzbach SD; Department of Biological Sciences, The University of Memphis, 103 Life Sciences Bldg, Memphis, TN, 38152, USA.
  • Skalli O; Department of Biological Sciences, The University of Memphis, 103 Life Sciences Bldg, Memphis, TN, 38152, USA. oskalli@memphis.edu.
Methods Mol Biol ; 1474: 217-32, 2016.
Article em En | MEDLINE | ID: mdl-27515083
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.
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Texto completo: 1 Base de dados: MEDLINE Assunto principal: Vimentina / Imuno-Histoquímica / Microscopia Imunoeletrônica / Inclusão do Tecido / Integrina beta3 / Células Endoteliais Idioma: En Ano de publicação: 2016 Tipo de documento: Article

Texto completo: 1 Base de dados: MEDLINE Assunto principal: Vimentina / Imuno-Histoquímica / Microscopia Imunoeletrônica / Inclusão do Tecido / Integrina beta3 / Células Endoteliais Idioma: En Ano de publicação: 2016 Tipo de documento: Article