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

La inmunocitoquímica (ICC, por sus siglas en inglés) y la inmunohistoquímica (IHC, por sus siglas en inglés) son técnicas de tinción que utilizan anticuerpos ligados a enzimas para visualizar y cuantificar proteínas o antígenos específicos en células cultivadas o secciones de tejido.
En ICC, se cultiva una sola capa de células objetivo en un cubreobjetos de vidrio. Las células se fijan con un agente de reticulación para evitar la degradación enzimática del antígeno, luego se tratan con un detergente para hacer que la membrana celular sea permeable.
En la IHQ, todo el tejido se fija y se incrusta en un bloque de parafina. Luego, la muestra se secciona en rodajas finas, que se pueden colocar en portaobjetos. Finalmente, se retira la parafina para recuperar el antígeno.
Una vez que los antígenos son accesibles en la muestra, se unen a anticuerpos primarios específicos del objetivo unidos con enzimas conjugadas como la peroxidasa de rábano picante.
La enzima cataliza la oxidación de la tinción DAB a un precipitado marrón, lo que permite la visualización de la proteína de interés.
Si la señal detectada es débil, se utilizan anticuerpos secundarios con enzimas conjugadas para unirse a un anticuerpo primario no marcado en la proteína diana para una mayor amplificación de la señal.

<ol>
	<li>Skoog, L. y Tani, E., 2011. Inmunocitoquímica: técnica indispensable en la citología rutinaria. Citopatología, 22(4), pp.215-229.</li>
	<li>Duraiyan, J., Govindarajan, R., Kaliyappan, K. y Palanisamy, M., 2012. Aplicaciones de la inmunohistoquímica. Revista de farmacia y ciencias bioafines, 4(Supl 2), p.S307.</li>
</ol>

inmunocitoquímica e inmunohistoquímica

Immunocytochemistry and Immunohistochemistry

La inmunocitoquímica (ICC) y la inmunohistoquímica (IHC) son técnicas que utilizan anticuerpos para detectar proteínas o antígenos específicos en una muestra. La técnica fue publicada por primera vez por Albert Coons en 1941 para detectar la presencia de antígeno neumocócico en secciones de tejido de ratones infectados con neumococo. La inmunocitoquímica ayuda a la localización de proteínas o antígenos en células individuales como células sanguíneas, células madre, etc., mientras que la inmunohistoquímica hace lo mismo para muestras de tejido.

Estas técnicas pueden ser colorimétricas si se utilizan anticuerpos conjugados con enzimas o si los anticuerpos marcados con fluoróforos basados ​​en fluorescencia localizan las proteínas o antígenos de interés. El producto coloreado obtenido a través de la reacción enzimática o la fluorescencia emitida por el fluoróforo se visualiza luego bajo un microscopio óptico o de inmunofluorescencia.

Estas técnicas pueden utilizar anticuerpos primarios o secundarios marcados con una enzima o un fluoróforo. Si se utiliza un anticuerpo primario, la técnica se denomina ICC directa o IHC, ya que los anticuerpos conjugados se unen directamente al epítopo de la proteína o antígeno de interés. En la técnica indirecta, un anticuerpo secundario marcado con la enzima o fluoróforo se une al anticuerpo primario unido al epítopo de la proteína o antígeno de interés. La señal emitida al unirse un anticuerpo secundario es más fuerte que la señal de un anticuerpo primario.

Ambas técnicas varían ligeramente en los pasos de preparación de la muestra. En la ICC, se genera una capa de monocultivo de células, que luego se tratan con agentes fijadores como formaldehído para evitar la degradación enzimática de los antígenos, seguido de un tratamiento con detergentes como Triton X o Tween 20 para hacer que la membrana celular sea permeable para la entrada de anticuerpos. De manera similar, en la IHC, las muestras de tejido se tratan primero con una solución fijadora, seguida de la inclusión en cera de parafina para evitar dañar el tejido frágil. Luego, las muestras de tejido se seccionan en rodajas finas y se tratan con detergente antes de la introducción de anticuerpos.

Una vez que las células o los portaobjetos de tejido se tratan con detergente, se añaden los anticuerpos. En la ICC o IHC directa, se añade el anticuerpo primario marcado con fluoróforo o enzimas y, después de la incubación, se visualizan con inmunofluorescencia o microscopio óptico. En la ICC o IHC indirecta, las muestras se incuban primero con el anticuerpo primario, seguido de un lavado para eliminar los anticuerpos no unidos. A continuación, se introduce el anticuerpo secundario, que se une al anticuerpo primario y emite fluorescencia o producto coloreado al añadir el colorante. Las muestras ya están listas para su visualización.

Inmunocitoquímica e inmunohistoquímica

La inmunocitoquímica (ICC) y la inmunohistoquímica (IHC) son técnicas que utilizan anticuerpos para detectar proteínas o antígenos específicos en una muestra. El...

immunocytochemistry-and-immunohistochemistry

Education

Educación

JoVE Core

Cell Biology

Biología Celular

Unit 1

Unidad 1

Visualizing Cells, Tissues, and Molecules

Visualización de células, tejidos y moléculas

KEY TERMS AND CONCEPTS

TÉRMINOS Y CONCEPTOS CLAVE

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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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...

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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 Biología Celular ilustra procesos celulares complejos mediante completos vídeos animados fáciles de entender. Esta colección se centra en los componentes esenciales de las células y su funcionamiento, incluyendo los orgánulos y las membranas, el transporte celular y el metabolismo energético celular. Todo este material se complementa con los vídeos de científicos en acción, que muestran la aplicación de la biología celular en experimentos reales de investigación.

Antibody Staining in Cells and Tissues

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