Name: immunogold labeling in electron microscopy
Uploaded: 2025-05-22T09:05:46.000Z
Duration: 1 min 20 s
Channel: JoVE

In immunogold electron microscopy, a target molecule is labeled using gold-conjugated antibodies.
The gold increases the electron scattering to give high-contrast dark spots. These spots can be distinguished from the other unlabelled structures and allow localization of the target molecule.
Direct immunolabeling uses a gold probe attached to a primary antibody that directly binds to the target molecule.&#160;
Indirect labeling uses a gold-labeled secondary antibody that binds to an unlabelled primary antibody. Indirect labeling is used more often due to its higher sensitivity.
Immunolabeling can be performed using several different techniques &#8212; post-embedding, pre-embedding, or whole-mount.
In the post-embedding technique, a sample is embedded in resin and then sliced into ultrathin sections that are then treated with gold-labeled antibodies.
In the pre-embedding technique, a sample is first incubated with the gold-labeled antibodies and then resin-embedded and cross-sectioned.
The whole-mount technique does not involve resin-embedding. Antibody reactions are carried out directly on a sample placed on the sample holder.

immunogold-elektronenmicroscopie

Immunogold Electron Microscopy

Immuno-elektronenmicroscopie maakt gebruik van immunogold-labeling van endogene eiwitten met specifieke antilichamen om deze eiwitten in cellen en weefsels te detecteren en te lokaliseren. De procedure biedt inzicht in de distributie en kwantificering van eiwitten onder verschillende stimulatieomstandigheden en biedt aanwijzingen over hun functies. Het conjugeren van zeer elektronendichte gouddeeltjes met primaire of secundaire antilichamen maakt antigeendetectie op en in cellen mogelijk, met hoge resolutie en specificiteit. Immuno-elektronenmicroscopie is gebruikt om de cellulaire en subcellulaire lokalisatie te identificeren van eiwitten die betrokken zijn bij neurotransmissie, nucleaire eiwitcomponenten en identificatie van immuunceltypen.

Er worden drie benaderingen toegepast om celantigenen te lokaliseren met behulp van transmissie-elektronenmicroscopie. Ten eerste omvatten de protocollen bij het bestuderen van de lokalisatie van intracellulaire antigenen antilichaamlabeling na inbedding in acrylharsen, antilichaamlabeling vooraf inbedden in de hars gecombineerd met celmembraanpermeabilisatie, of cryo-ultramicrotomie zonder inbedding. Ten tweede, wanneer celoppervlakte-eiwitten moeten worden gelokaliseerd, wordt het pre-inbeddingsprotocol gebruikt, waarbij labeling met immunogold-antilichamen wordt uitgevoerd voordat het in de hars wordt ingebed. Bij de derde techniek, de 'whole-mount'-techniek, is er geen sprake van het inbedden van hars, en worden de antilichaamreacties rechtstreeks op een monster uitgevoerd om de oppervlaktemoleculen te lokaliseren.

Voor scanning-elektronenmicroscopie moet het betreffende oppervlak noodzakelijkerwijs worden blootgesteld. Het monster kan direct worden gefixeerd en geëtiketteerd als het buitenoppervlak van het plasmamembraan van belang is. De cellen moeten echter worden gepermeabiliseerd met detergenten en immunologisch worden gemerkt voor het bestuderen van intracellulaire componenten of antigenen die in een weefsel zijn geïntegreerd. Voor weergave wordt het monster door vriesbreuk gebroken (snel invriezen en breken met een mes). Veel intracellulaire componenten, met name macromoleculaire complexen, zoals chromatine, eiwitcomplexen of virussen, kunnen worden geïsoleerd, gefixeerd en immunologisch gemerkt voor SEM. Ten slotte wordt een immunonegatieve kleuringstechniek toegepast voor oppervlakte-antigenen op kleine monsters, zoals virussen en bacteriën, die zich lenen voor negatieve kleuring.

Immunogold-elektronenmicroscopie

Immunoelectron microscopy utilizes immunogold labeling of endogenous proteins with specific antibodies to detect and localize these proteins in cells and ...

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immunogold labeling in electron microscopy

Immunogold labeling in electron microscopy uses antibodies and tiny gold particles to find specific proteins in cells and tissues. The gold particles are very electron-dense, which means they show up clearly in electron microscope images. This gives researchers a high-resolution way to see where proteins are located and to compare their amounts under different stimulation conditions.

The method can reveal proteins on the cell surface and inside cells. It has been used to study proteins involved in neurotransmission, parts of the nucleus, and markers for immune cell types. The same labeling idea can be paired with different sample-preparation methods, depending on what needs to be seen.

For transmission electron microscopy, three main approaches are used to localize cell antigens. Intracellular antigens can be labeled after embedding in acrylic resin, before embedding with membrane permeabilization, or by cryo-ultramicrotomy without embedding. When cell-surface proteins are the target, pre-embedding labeling is used so the immunogold antibodies bind before the sample is placed in resin.

A whole-mount technique is also used. In this method, the sample is not embedded in resin, and the antibody reaction happens directly on the specimen to identify surface molecules. For scanning electron microscopy, the surface of interest must be exposed. The sample can be fixed and labeled directly if the outer surface of the plasma membrane is being studied.

If intracellular components or antigens within a tissue are being examined with SEM, the cells must be permeabilized with detergents and then immunolabeled. The sample is then freeze-fractured, which means it is rapidly frozen and broken with a knife for viewing. Many intracellular components, including chromatin, protein complexes, and viruses, can be isolated, fixed, and immunolabeled this way.

An immunonegative staining technique is used for small specimens such as viruses and bacteria. This method is suited to surface antigens on samples that work well with negative staining. Together, these approaches let scientists localize proteins and other antigens with strong specificity and fine structural detail.

Immunogold Labeling in Electron Microscopy

immunogold-labeling-in-electron-microscopy

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immunogold electron microscopy

Immunogold labeling in electron microscopy uses antibodies and tiny gold particles to find specific proteins in cells and tissues. The gold particles are very electron-dense, which means they show up clearly in electron microscope images. This gives researchers a high-resolution way to see where proteins are located and to compare their amounts under different stimulation conditions. The method can reveal proteins on the cell surface and inside cells. It has been used to study proteins...

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key terms and concepts

Learn how immunogold labeling uses antibody-bound gold particles to map proteins in cells and tissues with transmission and scanning electron microscopy.

Immunogold Labeling in Electron Microscopy | JoVE K12

cell biology

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Immunogold Labeling in Electron Microscopy

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