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

イムノゴールド電子顕微鏡では、金標識抗体を用いて標的分子を標識します。
金は電子散乱を増加させて、コントラストの高いダークスポットを与えます。これらのスポットは、他の非標識構造と区別でき、標的分子の局在化を可能にします。
直接免疫標識は、標的分子に直接結合する一次抗体に結合した金プローブを使用します。 
間接標識では、非標識一次抗体に結合する金標識二次抗体を使用します。間接ラベリングは、感度が高いため、より頻繁に使用されます。
免疫標識は、ポストエンベッディング、プレエンベッド、またはホールマウントなど、いくつかの異なる手法を使用して実行できます。
ポストエンベッディング技術では、サンプルを樹脂に包埋し、極薄切片にスライスして、金標識抗体で処理します。
プレエンベッド法では、まずサンプルを金標識抗体とインキュベートし、次にレジンをエンベレントして断面化します。
ホールマウント技術は、樹脂の埋め込みを伴いません。抗体反応は、サンプルホルダーに置いたサンプル上で直接行われます。

免疫電子顕微鏡法

Immunogold Electron Microscopy

免疫電子顕微鏡法では、内因性タンパク質を特異的抗体で免疫金標識して、細胞および組織内のこれらのタンパク質を検出および位置発見します。 この手順により、さまざまな刺激条件下でのタンパク質の分布と定量化に関する洞察が得られ、タンパク質の機能に関する手がかりが得られます。 電子密度の高い金粒子を一次抗体または二次抗体と結合させることで、細胞上および細胞内の抗原を高い分解能と特異性で検出できます。 免疫電子顕微鏡法は、神経伝達に関与するタンパク質の細胞内および細胞内局在、核タンパク質成分の特定、および免疫細胞タイプの同定に使用されています。

透過型電子顕微鏡を使用して細胞抗原の位置を特定するために 3 つのアプローチが適用されます。 まず、細胞内抗原の局在を研究する場合、プロトコルには、アクリル樹脂への包埋後の抗体標識、細胞膜透過処理と組み合わせた樹脂への事前包埋抗体標識、または包埋を行わない凍結超切片術が含まれます。 第二に、細胞表面タンパク質の局在を特定する場合、樹脂に包埋する前に免疫金抗体による標識を行う前包埋プロトコルが使用されます。 3 番目の手法であるホールマウント手法では、樹脂の埋め込みを行わず、抗体反応をサンプル上で直接実行して表面分子の位置を発見します。

走査型電子顕微鏡では、対象となる表面を必ず露出させる必要があります。 細胞膜の外表面に関心がある場合は、サンプルを直接固定して標識することができます。 ただし、細胞内成分や組織に組み込まれた抗原を研究するには、細胞を界面活性剤で透過処理し、免疫標識する必要があります。 観察するために、サンプルは凍結破砕（急速に凍らせてナイフで割る）されます。 多くの細胞内成分、特にクロマチン、タンパク質複合体、ウイルスなどの高分子複合体を単離、固定し、SEM 用に免疫標識することができます。 最後に、免疫陰性染色技術が、陰性染色に適したウイルスや細菌などの小さな標本の表面抗原に適用されます。

免疫電子顕微鏡は、内因性タンパク質を特異的抗体で免疫金で標識し、これらのタンパク質を細胞内で検出し、局在化します。

Education

JoVE Core

Cell Biology

細胞生物学

Unit 1

ユニット1

Visualizing Cells, Tissues, and Molecules

細胞・組織・分子の可視化

KEY TERMS AND CONCEPTS

主要用語と概念

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 細胞生物学は、複雑な細胞プロセスを、わかりやすく包括的なアニメーション ビデオ レッスンで説明します。このコレクションは、細胞小器官や膜、細胞輸送、細胞のエネルギー学など、細胞の必須コンポーネントと機能に焦点を当てています。これは、実際の研究実験における細胞生物学の応用を実証する科学者による実際のビデオによって補足されています。

Immunogold Labeling in Electron Microscopy

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