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

Nella microscopia a immunofluorescenza, gli anticorpi marcati con fluorofori emettono fluorescenza dopo aver legato un bersaglio specifico o un antigene.
La microscopia a immunofluorescenza utilizza la luce per eccitare gli elettroni del fluoroforo a uno stato energetico più elevato. Quando tornano allo stato fondamentale, gli elettroni rilasciano una lunghezza d'onda della luce più lunga.
Questa emissione o fluorescenza consente la visualizzazione di cellule specifiche all'interno dei tessuti o di particolari proteine all'interno delle cellule.
L'immunofluorescenza può essere diretta quando l'anticorpo primario marcato con fluoroforo si lega alla proteina bersaglio. Oppure, può essere indiretto, in cui gli anticorpi secondari marcati con fluorofori legano uno specifico anticorpo primario attaccato alla proteina di interesse. La fluorescenza risultante è più forte di quella emessa dall'immunofluorescenza diretta.
L'immunofluorescenza diretta viene utilizzata per rilevare l'aggregazione proteica anomala nei tessuti, mentre l'immunofluorescenza indiretta può rilevare gli anticorpi circolanti nel siero durante la diagnosi di malattia autoimmune.

<ol>
	<li>Odell, I.D., e Cook, D., 2013. Tecniche di immunofluorescenza. Il giornale di dermatologia investigativa, 133(1), p.e4.</li>
	<li>Sanderson, M.J., Smith, I., Parker, I. e Bootman, M.D., 2014. Microscopia a fluorescenza. Protocolli del porto di Cold Spring, 2014(10), pp.pdb-top071795.</li>
</ol>

microscopia ad immunofluorescenza

Immunofluorescence Microscopy

Un microscopio a fluorescenza utilizza cromofori fluorescenti chiamati fluorocromi, che possono assorbire energia da una sorgente luminosa e quindi emettere questa energia come luce visibile. I fluorocromi includono sostanze naturalmente fluorescenti (come le clorofille) e coloranti fluorescenti che vengono aggiunti al campione per creare contrasto. Coloranti come il Texas Red e il FITC sono esempi di fluorocromi. Altri esempi includono i coloranti dell’acido nucleico 4’,6’-diamidino-2-fenilindolo (DAPI) e l’arancio di acridina.

Il microscopio irradia il campione con eccitazione a lunghezza d'onda corta, come la luce ultravioletta o blu. I cromofori assorbono la luce di eccitazione ed emettono luce visibile con lunghezze d'onda maggiori. La luce di eccitazione viene quindi filtrata (in parte perché la luce ultravioletta è dannosa per gli occhi) in modo che solo la luce visibile passi attraverso la lente oculare, producendo un'immagine del campione con colori vivaci su uno sfondo scuro.

I microscopi a fluorescenza possono identificare agenti patogeni, trovare specie particolari all'interno di un ambiente o trovare la posizione di particolari molecole e strutture all'interno di una cellula. Sono stati sviluppati anche approcci per distinguere le cellule vive da quelle morte in base al fatto che assorbono particolari fluorocromi. A volte, vengono utilizzati più fluorocromi sullo stesso campione per mostrare strutture o caratteristiche diverse.

Una delle applicazioni più importanti della microscopia a fluorescenza è l'immunofluorescenza, che viene utilizzata per identificare determinati microbi osservando se gli anticorpi si legano a loro. (Gli anticorpi sono molecole proteiche prodotte dal sistema immunitario che si attaccano a specifici agenti patogeni per ucciderli o inibirli). Questa tecnica ha due approcci: test di immunofluorescenza diretta (DFA) e test di immunofluorescenza indiretta (IFA). Nel DFA, gli anticorpi specifici (ad esempio, quelli che prendono di mira il virus della rabbia) sono colorati con un fluorocromo. Se il campione contiene l'agente patogeno bersaglio, è possibile osservare gli anticorpi che si legano all'agente patogeno al microscopio a fluorescenza. Questa è una colorazione anticorpale primaria perché gli anticorpi colorati si attaccano direttamente all'agente patogeno.

Nell'IFA, gli anticorpi secondari sono colorati con un fluorocromo anziché con gli anticorpi primari. Gli anticorpi secondari non si attaccano direttamente all'organismo ma si legano agli anticorpi primari. Quando gli anticorpi primari non colorati si legano al patogeno, si può osservare che gli anticorpi secondari fluorescenti si legano agli anticorpi primari. Pertanto, gli anticorpi secondari si attaccano indirettamente all'agente patogeno. Poiché più anticorpi secondari possono spesso attaccarsi a un anticorpo primario, l'IFA aumenta il numero di anticorpi fluorescenti attaccati al campione, facilitando la visualizzazione delle sue caratteristiche.

This text is adapted from Openstax, Microbiology 2e, Section 2.4: Staining Microscopic Specimens.

Microscopia ad immunofluorescenza

Un microscopio a fluorescenza utilizza cromofori fluorescenti chiamati fluorocromi, che possono assorbire energia da una sorgente luminosa e poi emettere questa energia ...

Education

Didattica

JoVE Core

Cell Biology

Biologia cellulare

Unit 1

Unità 1

Visualizing Cells, Tissues, and Molecules

Visualizzazione di cellule, tessuti e molecole

KEY TERMS AND CONCEPTS

TERMINI E CONCETTI CHIAVE

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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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

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Fluorescent Antibody Staining in Cells

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