Name: fluorescent antibody staining in cells
Uploaded: 2023-04-30T11:59:57.000Z
Duration: 1 min 12 s
Channel: JoVE

En la microscopía de inmunofluorescencia, los anticuerpos marcados con fluoróforos emiten fluorescencia al unirse a un objetivo específico o a un antígeno.
La microscopía de inmunofluorescencia utiliza luz para excitar los electrones del fluoróforo a un estado de energía más alto. A medida que regresan al estado fundamental, los electrones liberan una longitud de onda de luz más larga.
Esta emisión o fluorescencia permite la visualización de células específicas dentro de los tejidos o proteínas particulares dentro de las células.
La inmunofluorescencia puede ser directa cuando el anticuerpo primario marcado con fluoróforos se une a la proteína diana. O puede ser indirecto, donde los anticuerpos secundarios marcados con fluoróforos se unen a un anticuerpo primario específico unido a la proteína de interés. La fluorescencia resultante es más fuerte que la emitida por la inmunofluorescencia directa.
La inmunofluorescencia directa se utiliza para detectar la agregación anormal de proteínas en los tejidos, mientras que la inmunofluorescencia indirecta puede detectar los anticuerpos circulantes en el suero durante el diagnóstico de enfermedades autoinmunes.

<ol>
	<li>Odell, I.D., y Cook, D., 2013. Técnicas de inmunofluorescencia. Revista de dermatología investigativa, 133(1), p.e4.</li>
	<li>Sanderson, M.J., Smith, I., Parker, I. y Bootman, M.D., 2014. Microscopía de fluorescencia. Protocolos de Cold Spring Harbor, 2014(10), pp.pdb-top071795.</li>
</ol>

microscopía de inmunofluorescencia

Immunofluorescence Microscopy

Un microscopio de fluorescencia utiliza cromóforos fluorescentes llamados fluorocromos, que pueden absorber energía de una fuente de luz y entonces emitir esta energía como luz visible. Los fluorocromos incluyen sustancias naturalmente fluorescentes (como las clorofilas) y tintes fluorescentes que se agregan a la muestra para crear contraste. Tintes como el rojo Texas y el FITC son ejemplos de fluorocromos. Otros ejemplos incluyen los colorantes de ácido nucleico 4’,6’-diamidino-2-fenilindol (DAPI) y la naranja de acridina.

El microscopio irradia la muestra con excitación de longitud de onda corta, como luz ultravioleta o azul. Los cromóforos absorben la luz de excitación y emiten luz visible de longitudes de onda más largas. entonces, la luz de excitación se filtra (en parte porque la luz ultravioleta es perjudicial para los ojos) de modo que sólo la luz visible pasa a través de la lente ocular, produciendo una imagen de la muestra en colores brillantes sobre un fondo oscuro.

Los microscopios de fluorescencia pueden identificar patógenos, encontrar especies particulares dentro de un entorno o encontrar la ubicación de moléculas y estructuras particulares dentro de una célula. También se han desarrollado enfoques para distinguir las células vivas de las muertas en función de si absorben determinados fluorocromos. A veces, se utilizan varios fluorocromos en la misma muestra para mostrar diferentes estructuras o características.

Una de las aplicaciones más importantes de la microscopía de fluorescencia es la inmunofluorescencia, que se utiliza para identificar ciertos microbios observando si los anticuerpos se unen a ellos. (Los anticuerpos son moléculas de proteína que produce el sistema inmunológico y que se adhieren a patógenos específicos para matarlos o inhibirlos). Esta técnica tiene dos enfoques: ensayo de inmunofluorescencia directa (DFA) y ensayo de inmunofluorescencia indirecta (IFA). En DFA, los anticuerpos específicos (por ejemplo, los que se dirigen al virus de la rabia) se tiñen con un fluorocromo. Si la muestra contiene el patógeno objetivo, se pueden observar los anticuerpos que se unen al patógeno bajo el microscopio fluorescente. Esta es una tinción de anticuerpos primaria porque los anticuerpos teñidos se adhieren directamente al patógeno.

En IFA, los anticuerpos secundarios se tiñen con un fluorocromo en lugar de anticuerpos primarios. Los anticuerpos secundarios no se unen directamente al organismo sino que se unen a los anticuerpos primarios. Cuando los anticuerpos primarios no teñidos se unen al patógeno, se puede observar que los anticuerpos secundarios fluorescentes se unen a los anticuerpos primarios. Por tanto, los anticuerpos secundarios se unen indirectamente al patógeno. Dado que a menudo se pueden unir varios anticuerpos secundarios a un anticuerpo primario, la IFA aumenta la cantidad de anticuerpos fluorescentes adheridos a la muestra, lo que facilita la visualización de sus características.

Microscopía de inmunofluorescencia

Un microscopio de fluorescencia utiliza cromóforos fluorescentes llamados fluorocromos, que pueden absorber energía de una fuente de luz y luego emitir esta energía ...

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

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 DAPI, which stands for 4',6'-diamidino-2-phenylindole, and acridine orange. The sample is exposed to short-wavelength light, such as ultraviolet or blue light. The fluorochromes absorb this excitation light and emit light at a longer wavelength.

The microscope then filters out the excitation light. This is done partly because ultraviolet light can harm the eyes. Only the visible light reaches the ocular lens. That is why the specimen appears in bright colors on a dark background. Fluorescence microscopy can help identify pathogens, locate particular species in an environment, and show where molecules or structures are found inside a cell.

Some fluorescence methods can also help tell living cells from dead cells. They do this by using fluorochromes that are taken up differently by the cells. In some specimens, more than one fluorochrome is used at the same time. This lets scientists show different structures or features in the same sample.

A major application of this technique is immunofluorescence. Immunofluorescence identifies certain microbes by checking whether antibodies bind to them. Antibodies are protein molecules made by the immune system that attach to specific pathogens and help kill or block them. The method has two forms: direct immunofluorescence assay, or DFA, and indirect immunofluorescence assay, or IFA.

In DFA, the specific antibodies are stained with a fluorochrome before they are added to the specimen. For example, antibodies that target the rabies virus can be used this way. If the pathogen is present, the stained antibodies bind directly to it under the fluorescent microscope. This is called a primary antibody stain because the stained antibodies attach straight to the pathogen.

In IFA, the primary antibodies are not stained. Instead, the secondary antibodies carry the fluorochrome and bind to the primary antibodies. After the unstained primary antibodies attach to the pathogen, the fluorescent secondary antibodies can be seen attached to them. This makes the pathogen appear indirectly labeled. Because several secondary antibodies can bind to one primary antibody, IFA often produces a stronger fluorescent signal and makes the specimen easier to see.

Fluorescent Antibody Staining in Cells

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immunofluorescence microscopy

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...

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scanning electron microscopy

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key terms and concepts

Learn how fluorescence microscopes use fluorochromes and antibody staining to reveal pathogens, cells, and cell structures through direct and indirect immunofluorescence.

Fluorescent Antibody Staining in Cells | 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.

Fluorescent Antibody Staining in Cells

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