Immunogold labelling

Source: Wikipedia, the free encyclopedia.
mitochondria (B) mtDNA marked with gold particles after extraction.[1]

Immunogold labeling or immunogold staining (IGS) is a staining technique used in

antibodies which are in turn attached to primary antibodies designed to bind a specific antigen or other cell component. Gold is used for its high electron density which increases electron scatter to give high contrast 'dark spots'.[3]

First used in 1971, immunogold labeling has been applied to both

brightfield microscopy
. The labeling technique can be adapted to distinguish multiple objects by using differently-sized gold particles.

Immunogold labeling can introduce artifacts, as the gold particles reside some distance from the labelled object and very thin sectioning is required during sample preparation.[3]

History

Immunogold labeling was first used in 1971 by Faulk and Taylor to identify

antigens.[2][4] It was first applied in transmission electron microscopy (TEM) and was especially useful in highlighting proteins found in low densities, such as some cell surface antigens.[5] As the resolution of scanning electron microscopy (SEM) increased, so too did the need for nanoparticle-sized labels such as immunogold. In 1975, Horisberger and coworkers successfully visualised gold nanoparticles with a diameter of less than 30 nm[6]
and this soon became an established SEM technique.[5]

Technique

First, a thin section of the sample is cut, often using a microtome.[7] Various other stages of sample preparation may then take place.

The prepared sample is then incubated with a specific antibody designed to bind the molecule of interest.

Fc regions in a non-specific way.[6]

The electron-dense gold particle can now be seen under an electron microscope as a black dot, indirectly labeling the molecule of interest.[3]

Labeling multiple objects

Immunogold labeling can be used to visualize more than one target simultaneously. This can be achieved in

peptides.[9] A more complex method of multi-site labeling involves labeling opposite sides of an antigenic site separately, the immunogold particles attached to both sides can then be viewed simultaneously.[10]

Uses in brightfield microscopy

Although immunogold labeling is typically used for transmission electron microscopy, when the gold is 'silver-enhanced' it can be seen using

Erwinia amylovora.[11]

Limitations

An inherent limitation to the immunogold technique is that the gold particle is around 15-30 nm away from the site to which the primary antibody is bound[5] (when using a primary and secondary antibodies labeling strategy). The precise location of the targeted molecule can therefore not be accurately calculated. Gold particles can be created with a diameter of 1 nm (or lower) but another limitation is then realized—at these sizes the gold label becomes hard to distinguish from tissue structure.[2][5]

Thin sections are required for immunogold labeling and these can produce misleading images; a thin slice of a cell component may not give an accurate view of its

three-dimensional structure. For example, a microtubule may appear as a 'spike' depending on which plane the sectioning occurred. To overcome this limitation serial sections can be taken, which can then be compiled into a three-dimensional image.[3]

A further limitation is that antibodies and gold particles cannot penetrate the resin used to embed samples for imaging. Thus, only accessible molecules can be targeted and visualized. Labeling prior to embedding the sample can reduce the negative impact of this limitation.[3]

See also

References

  1. PMID 15157274
    .
  2. ^ a b c "Immunogold Labeling in Scanning Electron Microscopy". Archived from the original on 2014-02-06. Retrieved 2010-07-08.
  3. ^
    ISBN 978-0-8153-4106-2
    .
  4. PMID 4110101
    .
  5. ^
    PMID 8858365
    .
  6. ^
    PMID 366014
    .
  7. PMID 13124776
    .
  8. PMID 632554
    .
  9. PMID 6189888
    .
  10. PMID 6172469
    .
  11. ^
    PMID 2416717
    .
Retrieved from "https://en.wikipedia.org/w/index.php?title=Immunogold_labelling&oldid=1190914557"