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

Imunocitoquímica ou ICC e imuno-histoquímica ou IHC são técnicas de coloração que usam anticorpos ligados a enzimas para visualizar e quantificar proteínas ou antígenos específicos em células cultivadas ou seções de tecido.
No ICC, uma única camada de células-alvo é cultivada em uma lamínula de vidro. As células são fixadas com um agente de reticulação para evitar a degradação enzimática do antígeno e, em seguida, tratadas com um detergente para tornar a membrana celular permeável.
Na IHQ, todo o tecido é fixado e embebido em um bloco de parafina. Em seguida, a amostra é seccionada em fatias finas, que podem ser colocadas em lâminas. Finalmente, a parafina é removida para recuperar o antígeno.
Uma vez que os antígenos estão acessíveis na amostra, eles são ligados por anticorpos primários específicos do alvo ligados a enzimas conjugadas como a peroxidase de rábano.
A enzima catalisa a oxidação da coloração DAB para um precipitado marrom, permitindo a visualização da proteína de interesse.
Se o sinal detectado for fraco, anticorpos secundários com enzimas conjugadas são usados para ligar um anticorpo primário não marcado na proteína-alvo para amplificação adicional do sinal.

<ol>
	<li>Skoog, L. e Tani, E., 2011. Imunocitoquímica: uma técnica indispensável na citologia de rotina. Citopatologia, 22(4), pp.215-229.</li>
	<li>Duraiyan, J., Govindarajan, R., Kaliyappan, K. e Palanisamy, M., 2012. Aplicações de imuno-histoquímica. Jornal de farmácia e ciências bioaliadas, 4 (Suppl 2), p.S307.</li>
</ol>

imunocitoquímica e imunohistoquímica

Immunocytochemistry and Immunohistochemistry

A imunocitoquímica (ICQ) e a imunohistoquímica (IHQ) são técnicas que utilizam anticorpos para verificar proteínas ou antígenos específicos em uma amostra. A técnica foi publicada pela primeira vez por Albert Coons em 1941 para detectar a presença de antígeno pneumocócico em secções de tecido de camundongos infectados com Pneumococcus. A imunocitoquímica ajuda na localização de proteínas ou antígenos em células individuais, como células sanguíneas, células-tronco, etc., enquanto a imunohistoquímica faz o mesmo para amostras de tecido. Estas técnicas podem ser colorimétricas se forem utilizados anticorpos conjugados com enzimas, ou se anticorpos marcados com fluoróforo baseados em fluorescência localizarem as proteínas ou antígenos de interesse. O produto colorido obtido pela reação enzimática ou pela fluorescência emitida pelo fluoróforo é então visualizado em microscópio óptico ou de imunofluorescência. Estas técnicas podem utilizar anticorpos primários ou secundários marcados com uma enzima, ou um fluoróforo. Se for utilizado um anticorpo primário, a técnica é referida como ICQ ou IHQ direta, uma vez que os anticorpos conjugados se ligam diretamente ao epítopo da proteína ou antígeno de interesse. Indiretamente, um anticorpo secundário marcado com a enzima ou fluoróforo liga-se ao anticorpo primário ligado ao epítopo da proteína ou antígeno de interesse. O sinal emitido após a ligação de um anticorpo secundário é mais forte que o sinal de um anticorpo primário. Ambas as técnicas variam ligeiramente nas etapas de preparação da amostra. Na ICQ é gerada uma camada de monocultura de células, que são então tratadas com agentes fixadores como o formaldeído para evitar a degradação enzimática dos antígenos, seguida de tratamento com detergentes como Triton X ou Tween 20 para tornar a membrana celular permeável à entrada de anticorpos. Da mesma forma, na IHQ, as amostras de tecido são primeiro tratadas com uma solução fixadora, seguida de inclusão em parafina para evitar danos ao tecido frágil. As amostras de tecido são então seccionadas em fatias finas e tratadas com detergente antes da introdução dos anticorpos. Depois que as células ou lâminas de tecido são tratadas com detergente, os anticorpos são então adicionados. Na ICQ ou IHQ direta, o anticorpo primário marcado com fluoróforo, ou as enzimas, é adicionado e, após a incubação, são visualizados sob imunofluorescência, ou microscópio óptico. Na ICQ ou IHQ indireta, as amostras são primeiro incubadas com o anticorpo primário, seguidas de lavagem para remover os anticorpos não ligados. O anticorpo secundário é então introduzido, que se liga ao anticorpo primário e emite fluorescência ou produto colorido ao adicionar corante. As amostras agora estão prontas para visualização.

Imunocitoquímica e Imunohistoquímica

Imunocitoquímica (ICC) e imuno-histoquímica (IHQ) são técnicas que usam anticorpos para verificar proteínas ou antígenos específicos em uma amostra. O...

immunocytochemistry-and-immunohistochemistry

Education

JoVE Core

Cell Biology

Biologia Celular

Unit 1

Unidade 1

Visualizing Cells, Tissues, and Molecules

Visualizando Células, Tecidos e Moléculas

KEY TERMS AND CONCEPTS

TERMOS-CHAVE E CONCEITOS

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

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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

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Microscope Contrast for Live, Unstained Cells

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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

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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

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total internal reflection fluorescence microscopy

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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...

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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

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cryo-electron microscopy

Cryo-EM for Native Biomolecule Imaging

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electron microscope tomography and single-particle reconstruction

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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 - Biologia Celular ilustra processos celulares complexos em vídeos de animação fáceis de entender e, ao mesmo tempo, abrangentes. Esta coleção foca nos componentes e funções essenciais das células, incluindo organelas e membranas, transporte celular e energia celular. É complementado por vídeos de Cientistas em ação que demonstram a aplicação da biologia celular em experimentos de pesquisa do mundo real.

Antibody Staining in Cells and Tissues

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