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

L’immunocytochimie ou ICC et l’immunohistochimie ou IHC sont des techniques de coloration qui utilisent des anticorps liés à des enzymes pour visualiser et quantifier des protéines ou des antigènes spécifiques dans des cellules ou des coupes de tissus en culture.
Dans l’ICC, une seule couche de cellules ciblées est cultivée sur une lamelle de verre. Les cellules sont fixées à l’aide d’un agent de réticulation pour empêcher la dégradation enzymatique de l’antigène, puis traitées avec un détergent pour rendre la membrane cellulaire perméable.
Dans l’IHC, l’ensemble du tissu est fixé et noyé dans un bloc de paraffine. Ensuite, l’échantillon est sectionné en fines tranches, qui peuvent être placées sur des lames. Enfin, la paraffine est retirée pour récupérer l’antigène.
Une fois que les antigènes sont accessibles dans l’échantillon, ils sont liés par des anticorps primaires spécifiques à la cible liés à des enzymes conjuguées comme la peroxydase de raifort.
L’enzyme catalyse l’oxydation de la coloration DAB en un précipité brun, ce qui permet de visualiser la protéine d’intérêt.
Si le signal détecté est faible, des anticorps secondaires avec des enzymes conjuguées sont utilisés pour lier un anticorps primaire non marqué sur la protéine cible afin d’amplifier davantage le signal.

<ol>
	<li>Skoog, L. et Tani, E., 2011. L’immunocytochimie : une technique indispensable en cytologie de routine. Cytopathologie, 22(4), pp.215-229.</li>
	<li>Duraiyan, J., Govindarajan, R., Kaliyappan, K. et Palanisamy, M., 2012. Applications de l’immunohistochimie. Journal de pharmacie et de sciences biologiques, 4 (Suppl 2), p.S307.</li>
</ol>

immunocytochimie et immunohistochimie

Immunocytochemistry and Immunohistochemistry

L'immunocytochimie (ICC) et l'immunohistochimie (IHC) sont des techniques qui utilisent des anticorps pour rechercher des protéines ou des antigènes spécifiques dans un échantillon. La technique a été publiée pour la première fois par Albert Coons en 1941 pour détecter la présence d'antigène pneumococcique dans des coupes de tissus de souris infectées par Pneumocoque. L'immunocytochimie aide à la localisation de protéines ou d'antigènes dans des cellules individuelles comme les cellules sanguines, les cellules souches, etc., tandis que l'immunohistochimie fait de même pour les échantillons de tissus.

Ces techniques peuvent être colorimétriques si des anticorps conjugués à des enzymes sont utilisés ou si des anticorps marqués par un fluorophore basés sur la fluorescence localisent les protéines ou les antigènes d'intérêt. Le produit coloré obtenu via la réaction enzymatique ou la fluorescence émise par le fluorophore est ensuite visualisé au microscope optique ou à immunofluorescence.

Ces techniques peuvent utiliser des anticorps primaires ou secondaires marqués par une enzyme ou un fluorophore. Si un anticorps primaire est utilisé, la technique est appelée ICC ou IHC directe, car les anticorps conjugués se lient directement à l’épitope de la protéine ou de l’antigène d’intérêt. De manière indirecte, un anticorps secondaire marqué avec l'enzyme ou le fluorophore se lie à l'anticorps primaire attaché à l'épitope de la protéine ou de l'antigène d'intérêt. Le signal émis lors de la liaison d'un anticorps secondaire est plus fort que le signal d'un anticorps primaire.

Les deux techniques varient légèrement dans les étapes de préparation des échantillons. En ICC, une couche de monoculture de cellules est générée, qui est ensuite traitée avec des agents fixateurs comme le formaldéhyde pour empêcher la dégradation enzymatique des antigènes, suivie d'un traitement avec des détergents comme le Triton X ou le Tween 20 pour rendre la membrane cellulaire perméable à l'entrée de anticorps. De même, en IHC, les échantillons de tissus sont d’abord traités avec une solution de fixation, puis incorporés dans de la cire de paraffine pour éviter d’endommager les tissus fragiles. Les échantillons de tissus sont ensuite sectionnés en fines tranches et traités avec un détergent avant l'introduction des anticorps.

Une fois les cellules ou les lames de tissus traitées avec un détergent, les anticorps sont ensuite ajoutés. En ICC ou IHC direct, l'anticorps primaire marqué avec un fluorophore ou des enzymes est ajouté, et après incubation, ils sont visualisés sous immunofluorescence ou au microscope optique. En ICC ou IHC indirect, les échantillons sont d’abord incubés avec l’anticorps primaire, suivis d’un lavage pour éliminer les anticorps non liés. L'anticorps secondaire est ensuite introduit, qui se lie à l'anticorps primaire et émet une fluorescence ou un produit coloré lors de l'ajout du colorant. Les échantillons sont maintenant prêts à être visualisés.

Immunocytochimie et Immunohistochimie

L’immunocytochimie (ICC) et l’immunohistochimie (IHC) sont des techniques qui utilisent des anticorps pour vérifier la présence de protéines ou d’antigènes spécifiques dans un échantillon. Le...

immunocytochemistry-and-immunohistochemistry

Education

Enseignement

JoVE Core

Cell Biology

Biologie cellulaire

Unit 1

Unité 1

Visualizing Cells, Tissues, and Molecules

Visualisation des cellules, des tissus et des molécules

KEY TERMS AND CONCEPTS

TERMES ET CONCEPTS CLÉS

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

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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...

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Electron Microscopy Sample Prep Methods

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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 Biologie cellulaire illustre les processus cellulaires complexes dans des leçons vidéos complètes mais faciles à comprendre. Cette collection se concentre sur les composants et fonctions essentiels de la cellule ; cela inclut les organelles et les membranes ainsi que le transport et le métabolisme énergétique cellulaire. Elle est complétée par nos vidéos de scientifiques à l'action qui démontrent l'application de la biologie cellulaire dans des expériences concrètes.

Antibody Staining in Cells and Tissues

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