Double-label immunoelectron microscopy for studying the colocalization of proteins in cultured cells

Methods Mol Biol. 2010;657:249-57. doi: 10.1007/978-1-60761-783-9_20.

Abstract

Multiple label immunoelectron microscopy localizes and detects multiple antigens in cells and tissues. In double labeling, two kinds of primary antibodies from different animal species are used after being mixed in a single solution. To distinguish the different antigens, secondary antibodies should be labeled with colloidal gold particles of different diameter. Generally, the secondary antibody that is used for detecting the antigen with lower distribution density is labeled with smaller-sized gold particles. In this chapter, double-label immunoelectron microscopy of gelatin-embedded cultured cells using the cryosectioning technique is described.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibodies / chemistry
  • Antibodies / immunology
  • Antigens / analysis
  • Antigens / immunology
  • Caveolin 1 / analysis
  • Caveolin 1 / immunology
  • Cells, Cultured
  • Cryoultramicrotomy
  • Endothelial Cells / cytology
  • Endothelial Cells / immunology
  • Endothelial Cells / ultrastructure
  • Gelatin / chemistry
  • Gold Colloid / chemistry
  • Gold Colloid / immunology
  • Humans
  • Immunohistochemistry / methods*
  • Microscopy, Immunoelectron / methods*
  • Particle Size
  • Staining and Labeling / methods*
  • Tissue Embedding
  • rab5 GTP-Binding Proteins / analysis
  • rab5 GTP-Binding Proteins / immunology

Substances

  • Antibodies
  • Antigens
  • CAV1 protein, human
  • Caveolin 1
  • Gold Colloid
  • Gelatin
  • rab5 GTP-Binding Proteins