Immuno‐electron Microscopy

Abstract

Immuno‐electron microscopy combines the specificity and sensitivity of immunological methods with the high structural resolution of electron microscopy, providing an in situ correlation and visualization of antigen‐binding sites and ultrastructure.

Keywords: particulate markers; colloidal gold; chemical fixation; cryofixation; on‐section labelling

Figure 1.

Direct imaging of monovalent antibody fragments (Fab) bound to a T‐even bacteriophage by transmission electron microscopy. Fab molecules (arrowheads) against phage Tu‐6 allow the detection of homologous epitopes at distinct sites on the tail fibres of the related phage Tu‐46. The phage preparation was negatively stained with uranyl acetate. Bar, 0.1 μm.

Figure 2.

Indirect imaging of the viral glycoprotein gp70 on the surface of a Friend murine leukaemia virus. The picture shows an ultrathin section of a ferritin‐labelled virus particle in the extracellular space. The sample was fixed chemically and labelled in two steps, first with an antibody against gp70, followed by a secondary antibody against the primary antibody. The secondary antibody itself is conjugated to the electron‐dense marker molecule ferritin. In the centre of the image, the central viral core can be seen inside the envelope. The dark dots surrounding the virus are the iron‐containing cores of the ferritin particles. The distance between the surface of the viral particle and the ferritin cores reflects the space taken up by the primary and secondary antibodies as well as the protein shell of the ferritin molecule. This layer can be distinguished from the protein‐free extracellular space as a faintly grey cloud surrounding the virus. Bar, 0.1 μm.

Figure 3.

Replica of various stages of measles virus assembly on the surface of HeLa cells. Infected cells were fixed chemically and labelled with primary antibodies against viral haemagglutinin. The primary antibody was visualized by protein A conjugated to 10‐nm gold particles. After labelling, the sample was critical point dried and shadowed with a platinum–carbon layer. At all stages, viral particles can be identified by the presence of gold particles bound to the viral haemagglutinin. Viral preforms (patches and strands) can be seen on the cell surface, whereas the later stages of virus budding are located at the tips of membranous protrusions. The immuno‐gold surface replica technique allows direct correlation of the distribution of labelled surface antigens with the surface morphology of large areas. Bar, 0.5 μm.

Figure 4.

Cryosection of J774 mouse macrophages containing phagosomes with internalized 1‐μm latex beads. A thawed cryosection prepared according to the method of Tokuyasu was labelled with antibodies against filamentous F‐actin and protein A–10‐nm gold conjugates. Bar, 0.5 μm. (Micrograph kindly provided by Anja Habermann and Gareth Griffiths, European Molecular Biology Laboratory, Heidelberg, Germany.)

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Further Reading

Echlin P (1992) Low‐temperature Microscopy and Analysis. New York: Plenum Press.

Griffiths G (1993) Fine Structure Immunocytochemistry. Berlin: Springer.

Larsson L‐I (1988) Immunocytochemistry: Theory and Practice. Boca Raton, FL: CRC Press.

Skepper JN (2000) Immunocytochemical strategies for electron microscopy: choice or compromise. Journal of Microscopy 199: 1–36.

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How to Cite close
Schwarz, Heinz, and Hohenberg, Heinz(Apr 2001) Immuno‐electron Microscopy. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1038/npg.els.0003000]