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

В иммунозолотой электронной микроскопии молекула-мишень помечается с помощью антител, конъюгированных с золотом.
Золото увеличивает рассеяние электронов, давая высококонтрастные темные пятна. Эти пятна можно отличить от других немеченых структур, и они позволяют локализовать молекулу-мишень.
При прямом иммуномечении используется золотой зонд, прикрепленный к первичному антителу, которое напрямую связывается с молекулой-мишенью. 
При непрямом мечении используется вторичное антитело, меченное золотом, которое связывается с немеченным первичным антителом. Непрямая маркировка используется чаще из-за ее более высокой чувствительности.
Иммуномечение может быть выполнено с использованием нескольких различных методов — после встраивания, предварительного встраивания или целиком.
В технике после встраивания образец встраивается в смолу, а затем нарезается на ультратонкие срезы, которые затем обрабатываются антителами, меченными золотом.
В технике предварительного встраивания образец сначала инкубируют с антителами, меченными золотом, а затем засаживают смолой и делают поперечное сечение.
Метод цельного монтажа не включает в себя встраивание смолы. Реакции антител проводятся непосредственно на образце, помещенном на держатель образца.

Иммунозолото Электронная Микроскопия

Immunogold Electron Microscopy

Иммуноэлектронная микроскопия использует иммуно-золотое мечение эндогенных белков с помощью специфических антител для обнаружения и локализации этих белков в клетках и тканях. Процедура дает представление о распределении и количественном определении белков в различных условиях стимуляции, предлагая подсказки об их функциях. Сопряжение частиц золота с высокой электронной плотностью с первичными или вторичными антителами позволяет обнаруживать антигены на клетках и внутри них с высоким разрешением и специфичностью. Иммуноэлектронная микроскопия используется для выявления клеточной и субклеточной локализации белков, участвующих в нейротрансмиссии, компонентов ядерных белков, а также идентификации типов иммунных клеток. 

 Для локализации клеточных антигенов с помощью трансмиссионной электронной микроскопии применяются три подхода. Во-первых, при изучении локализации внутриклеточных антигенов протоколы включают мечение антител после внедрения в акриловые смолы, предварительное внедрение мечения антител в смолу в сочетании с пермеабилизацией клеточной мембраны или криоультрамикротомию без внедрения. Во-вторых, когда необходимо локализовать белки клеточной поверхности, используется протокол предварительного внедрения, при котором маркировка антителами иммунозолота выполняется перед внедрением в смолу. Третий метод, метод цельного монтирования, не предполагает заливки смолы, а реакции антител проводятся непосредственно на образце для обнаружения поверхностных молекул. 

 Для сканирующей электронной микроскопии интересующая поверхность обязательно должна быть экспонирована. Образец можно напрямую зафиксировать и пометить, если представляет интерес внешняя поверхность плазматической мембраны. Однако клетки должны быть проницаемы детергентами и иммуномаркированы для изучения внутриклеточных компонентов или антигенов, интегрированных в ткань. Для просмотра образец подвергается замораживанию (быстрому замораживанию и разрушению ножом). Многие внутриклеточные компоненты, особенно макромолекулярные комплексы, такие как хроматин, белковые комплексы или вирусы, можно выделить, зафиксировать и иммуномаркировать для СЭМ. Наконец, метод иммунонегативного окрашивания применяется для поверхностных антигенов небольших образцов, таких как вирусы и бактерии, которые поддаются негативному окрашиванию.

 <h4>Immunogold Electron Microscopy</h4>

 Immunoelectron microscopy utilizes immunogold labeling of endogenous proteins with specific antibodies to detect and localize these proteins in cells and tissues. The procedure provides insights into the distribution and quantification of protein under different stimulation conditions offering clues about their functions. Conjugating highly electron-dense gold particles with primary or secondary antibodies allow antigen detection on and within cells, with high resolution and specificity. Immunoelectron microscopy has been used to identify the cellular and subcellular localization of proteins involved in neurotransmission, nuclear protein components, and identification of immune cell types.

 Three approaches are applied to localize cell antigens using transmission electron microscopy. First, when studying the localization of intracellular antigens, protocols involve antibody labeling post-embedding in acrylic resins, antibody labeling pre-embedding in the resin combined with cell membrane permeabilization, or cryo-ultramicrotomy without embedding. Second, when cell-surface proteins are to be localized, the pre-embedding protocol is used where labeling with immunogold antibodies is done before embedding in the resin. The third technique, the whole-mount technique, does not involve resin-embedding, and the antibody reactions are carried out directly on a sample to locate the surface molecules.

 For scanning electron microscopy, the surface of interest must necessarily be exposed. The sample can be directly fixed and labeled if the outer surface of the plasma membrane is of interest. However, the cells must be permeabilized with detergents and immunolabeled for studying intracellular components or antigens integrated into a tissue. For viewing, the sample is freeze-fractured (rapid freezing and breaking with a knife). Many intracellular components, particularly macromolecular complexes, like chromatin, protein complexes, or viruses, can be isolated, fixed, and immunolabeled for SEM. Finally, an immunonegative staining technique is applied for surface antigens on small specimens, such as viruses and bacteria, which lend themselves to negative staining.

Иммуноэлектронная микроскопия использует мечение иммунозолотом эндогенных белков специфическими антителами для обнаружения и локализации этих белков в клетках и ...

Education

JoVE Core

Cell Biology

Клеточная биология

Unit 1

Раздел 1

Visualizing Cells, Tissues, and Molecules

Визуализация клеток, тканей и молекул

KEY TERMS AND CONCEPTS

ОСНОВНЫЕ ТЕРМИНЫ И ПОНЯТИЯ

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

imaging biological samples with optical microscopy

imaging-biological-samples-with-optical-microscopy

How Light Microscopes Bring Samples Into View

Optical microscopy uses light and lenses to make tiny biological samples visible. Antonie van Leeuwenhoek built the first compound optical microscope in the 17th century to view blood cells, bacteria, and yeast cells. In 1830, Joseph Jackson Lister developed an essentially modern light microscope. During the 20th century, microscopes gained better magnification and resolution. A sample is placed on a glass slide and clipped onto the stage, which is the flat platform of the microscope. The...

phase contrast and differential interference contrast microscopy

phase-contrast-and-differential-interference-contrast-microscopy

Microscope Contrast for Live, Unstained Cells

Phase-contrast and differential interference contrast microscopy help scientists see live cells without stains. Both methods boost contrast so thin, transparent structures become easier to study under the microscope. Phase-contrast microscopes use interference between light that passes straight through a cell and light that is refracted by cell parts. The microscope uses an annular stop in the condenser to make a hollow cone of light. The objective lens also contains a phase plate with a phase...

fixation and sectioning

Microscope Slide Prep and Staining

Microscope slide preparation includes wet mounts, fixation, and staining. These steps help make specimens easier to see under a light microscope. They also help preserve cells and highlight important structures. A wet mount is the simplest slide preparation. The specimen is placed in a drop of liquid on the slide. A liquid sample can be added directly with a dropper, and a solid sample, such as a skin scraping, can be placed on the slide before the liquid is added. The liquid is sometimes...

immunofluorescence microscopy

Fluorescent Antibody Staining in Cells

Fluorescent antibody staining uses a fluorescence microscope to make cells and microbes visible in bright colors against a dark background. The microscope works with fluorochromes, which are fluorescent dyes that absorb energy from a light source and then give off visible light. Some fluorochromes are natural, such as chlorophylls. Others are added to the specimen to improve contrast. Common examples of fluorochromes include Texas red and FITC. The method can also use nucleic acid dyes such as...

immunocytochemistry and immunohistochemistry

immunocytochemistry-and-immunohistochemistry

Antibody Staining in Cells and Tissues

Immunocytochemistry (ICC) and immunohistochemistry (IHC) use antibodies to detect specific proteins or antigens in samples. ICC focuses on individual cells, such as blood cells and stem cells. IHC is used for tissue samples. These methods were first published by Albert Coons in 1941, when they were used to detect pneumococcal antigen in mouse tissue sections from mice infected with Pneumococcus. Both techniques can produce either a colored signal or a fluorescent signal. Enzyme-conjugated...

confocal fluorescence microscopy

Confocal Microscopy for Sharp Cell Images

Confocal microscopy is an advanced imaging method used to capture sharp fluorescence images. Its main advantage is that it blocks out-of-focus light from the sample with pinholes. That helps produce high-resolution images with strong contrast. Unlike a standard optical microscope, a confocal microscope uses a focused laser beam to scan the sample surface at different z-planes. This makes it especially useful for thick specimens, such as biofilms. These samples can often be examined alive and...

protein dynamics in living cells

Fluorescent Methods for Protein Movement

Protein movement in living cells can be measured with fluorescence-based methods such as FRAP, FRET, and PET. These techniques help scientists track how proteins move, interact, and change distance inside the cell. Fluorescent recovery after photobleaching, or FRAP, measures protein movement rates within the cell. In this method, a small region is exposed to a strong laser beam. The beam permanently bleaches fluorophore-tagged proteins in that area. Over time, bleached proteins diffuse away...

total internal reflection fluorescence microscopy

total-internal-reflection-fluorescence-microscopy

Surface-Only Imaging with TIRF Microscopy

Total internal reflection fluorescence microscopy, or TIRF, is a microscope method used to view fluorophores near a solid surface with a higher refractive index, such as a glass coverslip. It is useful when scientists want to study signals close to the cell surface. TIRF works when light from a medium with a lower refractive index, such as air, hits the glass coverslip at a critical angle. Instead of entering the glass, the light undergoes total internal reflection. This reflection creates an...

atomic force microscopy

Atomic Force Microscopy for Surface Mapping

Atomic force microscopy is used to map the surface details of materials. It can examine ceramics, glass, polymers, and biological samples. The method gives far more resolution than an optical imaging system and produces three-dimensional surface profiles instead of flat 2D images. The probe is the key part of an AFM setup. It includes the cantilever and tip assembly, and it is also the part that is replaced most often. Constant contact with samples wears down the tip, so probe choice depends...

super-resolution fluorescence microscopy

Sharper Cell Images with Fluorescence Microscopy

Super-resolution fluorescence microscopy gives sharper images than standard fluorescence microscopy by reducing the point spread function, or PSF. The PSF is the light pattern from a point source that makes it look blurred. Because of this blur, each fluorescent spot appears larger than it really is. Nearby fluorophores can also overlap through their PSFs and make the image less clear. Several super-resolution methods have been developed to improve image detail. Photoactivated localization...

overview of electron microscopy

Electron Microscopy: TEM vs SEM

Electron microscopy uses electrons instead of visible light to make images with much higher detail than a light microscope. Visible light limits the resolution of light microscopes, which usually magnify up to 1000X, and sometimes up to 1500X. Electrons have a much shorter wavelength, about 0.005 nm, so they can produce resolution as fine as 0.05 nm. An electron microscope can also magnify images up to 2,000,000X and reveal subcellular structures and some molecular structures, including single...

scanning electron microscopy

How SEM Reveals Surface Details

A scanning electron microscope (SEM) reveals the surface details of a sample with an electron beam. The beam scans across the sample in a two-dimensional pattern. SEM can image areas from about 1 centimeter down to 5 micrometers wide. SEM is useful for studying bacteria, viruses, tissues, and larger samples such as insects. Conventional SEM can produce magnification from 20X to 30,000X. It also offers spatial resolution of about 50 to 100 nanometers. The process starts when the electron gun...

transmission electron microscopy

How TEM Reveals Tiny Structures

Transmission electron microscopy uses electrons to reveal very small structures inside a specimen. The technique grew from the first electron microscope prototype developed in 1931 by physicist Ernst Ruska. He built on the idea that magnetic fields can direct an electron beam, just as lenses direct light in an optical microscope. In a transmission electron microscope, a hot tungsten element produces electrons. An electron gun then accelerates them with a potential difference, giving the beam...

preparation of samples for electron microscopy

preparation-of-samples-for-electron-microscopy

Electron Microscopy Sample Prep Methods

Electron microscopy sample prep methods help biological samples survive imaging in TEM and SEM. Before a specimen can be viewed, it must be fixed, or stabilized, so the electron beam does not damage it. It also must be dried well, or desiccated, because the microscope uses a vacuum. Fixation should happen as quickly as possible. Once a sample leaves its natural environment, its properties begin to change. In tissue samples, oxygen levels can drop and change the appearance of mitochondria. That...

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...

cryo-electron microscopy

Cryo-EM for Native Biomolecule Imaging

Cryo-EM, or cryo-electron microscopy, lets scientists image biomolecules in their frozen, native state. It avoids the dehydration, fixation, and staining used in conventional electron microscopy, which can distort samples and create artifacts. It also reduces damage from the electron beam by using gentler imaging conditions. This method is useful when other structure tools have limits. X-ray diffraction needs a molecule to form a crystal, and that is not always possible. Even when...

electron microscope tomography and single-particle reconstruction

electron-microscope-tomography-and-single-particle-reconstruction

3D Imaging with Electron Microscopy

Transmission electron microscopy (TEM) can build 3D images of biological samples. It does this with electron microscope tomography and single-particle reconstruction. Tomography can show cell parts or small cells in vivo, while single-particle reconstruction is used for macromolecules and macromolecular complexes in vitro only. Electron tomography can be done in TEM or in STEM, which stands for scanning transmission electron microscopy. STEM is mainly used for thick biological specimens. It...

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

JoVE Core Cell Biology illustrates complex cellular processes in easy-to-understand yet comprehensive animated video lessons. This collection focuses on the essential components and functions of cells, including organelles and membranes, cellular transport, and cell energetics. It is complemented by scientist-in-action videos that demonstrate the application of cell biology in real-world research experiments.

Immunogold Labeling in Electron Microscopy

Description

Transcript