Name: antibody staining in cells and tissues
Uploaded: 2023-04-30T12:00:05.000Z
Duration: 1 min 22 s
Channel: JoVE

면역세포화학(ICC) 및 면역조직화학(IHC)은 효소 결합 항체를 사용하여 배양된 세포 또는 조직 절편에서 특정 단백질 또는 항원을 시각화하고 정량화하는 염색 기술입니다.
ICC에서는 표적 세포의 단일 층이 유리 커버슬립에서 성장합니다. 세포는 항원의 효소 분해를 방지하기 위해 가교제로 고정된 다음 세포막을 투과할 수 있도록 세제로 처리합니다.
IHC에서는 전체 조직이 파라핀 블록에 고정되어 박혀 있습니다. 그런 다음 샘플을 슬라이드에 배치할 수 있는 얇은 조각으로 절단합니다. 마지막으로, 파라핀을 제거하여 항원을 회수합니다.
항원이 샘플에서 접근할 수 있게 되면 양고추냉이 과산화효소(horseradish peroxidase)와 같은 접합 효소와 연결된 표적 특이적 1차 항체에 결합됩니다.
이 효소는 DAB 염색의 산화를 갈색 침전물로 촉매하여 관심 단백질을 시각화할 수 있습니다.
검출된 신호가 약한 경우, 접합체 효소가 포함된 2차 항체를 사용하여 추가 신호 증폭을 위해 표적 단백질에 표지되지 않은 1차 항체를 결합합니다.

<ol>
	<li>Skoog, L. 및 Tani, E., 2011. 면역세포화학(Immunocytochemistry): 일상적인 세포학에서 없어서는 안 될 기술. 세포병리학, 22(4), pp.215-229.</li>
	<li>Duraiyan, J., Govindarajan, R., Kaliyappan, K. 및 Palanisamy, M., 2012. 면역조직화학의 응용. Journal of pharmacy & bioallied sciences, 4(Suppl 2), p.S307.</li>
</ol>

면역세포화학 및 면역조직화학

Immunocytochemistry and Immunohistochemistry

면역세포화학(ICC) 및 면역조직화학(IHC)은 항체를 사용하여 검체 내 특정 단백질이나 항원을 확인하는 기술입니다. 이 기술은 폐렴구균에 감염된 쥐의 조직 절편에서 폐렴구균 항원의 존재를 검출하기 위해 1941년 앨버트 쿤스에 의해 처음 발표되었습니다. 면역세포화학은 혈액 세포, 줄기 세포 등과 같은 개별 세포에서 단백질이나 항원의 위치를 파악하는 데 도움이 되는 반면, 면역조직화학은 조직 샘플에 대해서도 동일한 역할을 합니다.

이러한 기술은 효소 결합 항체가 사용되거나 형광 기반 형광단 태그 항체가 관심 있는 단백질 또는 항원을 국소화하는 경우 비색적일 수 있습니다. 효소 반응이나 형광단에 의해 방출된 형광을 통해 얻은 착색된 생성물은 광학 또는 면역형광 현미경으로 시각화됩니다.

이러한 기술은 효소 또는 형광단으로 태그가 지정된 1차 또는 2차 항체를 사용할 수 있습니다. 1차 항체를 사용하는 경우 접합된 항체가 관심 단백질 또는 항원의 에피토프에 직접 결합하므로 이 기술을 직접 ICC 또는 IHC라고 합니다. 간접적으로, 효소 또는 형광단으로 태그된 2차 항체는 관심 단백질 또는 항원의 에피토프에 부착된 1차 항체에 결합합니다. 2차 항체가 결합할 때 방출되는 신호는 1차 항체의 신호보다 더 강합니다.

두 기술 모두 샘플 준비 단계에서 약간 다릅니다. ICC에서는 세포의 단일 배양 층이 생성되고 포름알데히드와 같은 고정제로 처리되어 항원의 효소 분해를 방지한 다음 Triton X 또는 Tween 20과 같은 세제로 처리하여 세포막이 침투할 수 있도록 만듭니다. 항체. 마찬가지로 IHC에서는 조직 샘플을 먼저 고정 용액으로 처리한 후 파라핀 왁스에 묻혀 연약한 조직의 손상을 방지합니다. 그런 다음 조직 샘플을 얇은 조각으로 나누고 항체를 도입하기 전에 세제로 처리합니다.

세포 또는 조직 슬라이드가 세제로 처리되면 항체가 추가됩니다. 직접 ICC 또는 IHC에서는 형광단 또는 효소로 태그된 1차 항체를 추가하고 배양 후 면역형광 또는 광학 현미경으로 시각화합니다. 간접 ICC 또는 IHC에서는 샘플을 먼저 1차 항체와 함께 배양한 다음 세척하여 결합되지 않은 항체를 제거합니다. 이후 2차 항체가 도입되는데, 이는 1차 항체와 결합하여 염료를 첨가하면 형광이나 착색 생성물을 방출합니다. 이제 샘플을 시각화할 준비가 되었습니다.

면역세포화학(ICC) 및 면역조직화학(IHC)은 항체를 사용하여 샘플에서 특정 단백질 또는 항원을 확인하는 기술입니다. 그만큼 ...

immunocytochemistry-and-immunohistochemistry

Education

교육

JoVE Core

Cell Biology

세포생물학

Unit 1

제1단원

Visualizing Cells, Tissues, and Molecules

세포, 조직 및 분자의 시각화

KEY TERMS AND CONCEPTS

주요 용어 및 개념

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 antibodies create a colorimetric result through a chemical reaction. Fluorophore-tagged antibodies produce fluorescence that can be seen with an immunofluorescence microscope. The signal can come from a primary antibody or from a secondary antibody, depending on the method used.

In direct ICC or IHC, the primary antibody is tagged with an enzyme or fluorophore and binds directly to the epitope, or target site, on the protein or antigen. In indirect ICC or IHC, an unlabeled primary antibody binds first, and then a tagged secondary antibody binds to it. The indirect method usually gives a stronger signal than the direct method.

The sample preparation steps are slightly different for cells and tissues. In ICC, a monoculture layer of cells is first fixed with a solution such as formaldehyde to prevent antigen breakdown. The cells are then treated with detergents like Triton X or Tween 20 so antibodies can enter the cells. In IHC, tissue samples are first fixed, then embedded in paraffin wax to protect fragile tissue. The tissue is cut into thin slices and treated with detergent before antibodies are added.

After detergent treatment, the antibodies are introduced. In direct ICC or IHC, the tagged primary antibody is added and the sample is later viewed under an optical or immunofluorescence microscope. In indirect ICC or IHC, the sample is first incubated with the primary antibody and washed to remove any unbound antibodies. The secondary antibody is then added, and it produces fluorescence or a colored product after dye addition. The slide is then ready for visualization.

Antibody Staining in Cells and Tissues

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 (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

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 ICC and IHC use tagged antibodies to detect proteins in cells and tissues, using color or fluorescence for microscope viewing.

Antibody Staining in Cells and Tissues | 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.

Antibody Staining in Cells and Tissues

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