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

W mikroskopii immunofluorescencyjnej przeciwciała znakowane fluoroforem emitują fluorescencję po związaniu określonego celu lub antygenu.
Mikroskopia immunofluorescencyjna wykorzystuje światło do wzbudzenia elektronów fluoroforu do wyższego stanu energetycznego. Gdy powracają do stanu podstawowego, elektrony uwalniają dłuższą długość fali światła.
Ta emisja lub fluorescencja umożliwia wizualizację określonych komórek w tkankach lub określonych białek w komórkach.
Immunofluorescencja może być bezpośrednia, gdy pierwszorzędowe przeciwciało znakowane fluoroforem wiąże się z białkiem docelowym. Lub może być pośredni, gdzie przeciwciała drugorzędowe oznaczone fluoroforami wiążą specyficzne przeciwciało pierwszorzędowe przyłączone do białka będącego przedmiotem zainteresowania. Uzyskana fluorescencja jest silniejsza niż ta emitowana przez immunofluorescencję bezpośrednią.
Immunofluorescencja bezpośrednia służy do wykrywania nieprawidłowej agregacji białek w tkankach, podczas gdy immunofluorescencja pośrednia może wykrywać przeciwciała krążące w surowicy podczas diagnozy choroby autoimmunologicznej.

<ol>
	<li>Odell, ID i Cook, D., 2013. Techniki immunofluorescencji. Dziennik dermatologii śledczej, 133(1), p.e4.</li>
	<li>Sanderson, M.J., Smith, I., Parker, I. i Bootman, M.D., 2014. Mikroskopia fluorescencyjna. Protokoły Cold Spring Harbor, 2014(10), s. pdb-top071795.</li>
</ol>

mikroskopia immunofluorescencyjna

Immunofluorescence Microscopy

Mikroskop fluorescencyjny wykorzystuje fluorescencyjne chromofory zwane fluorochromami, które mogą absorbować energię ze źródła światła, a następnie emitować tę energię w postaci światła widzialnego. Fluorochromy obejmują naturalnie fluorescencyjne substancje (takie jak chlorofile) i barwniki fluorescencyjne dodawane do próbki w celu uzyskania kontrastu. Przykładami fluorochromów są barwniki, takie jak czerwień teksańska i FITC. Inne przykłady obejmują barwniki kwasów nukleinowych 4’,6’-diamidyno-2-fenyloindol (DAPI) i oranż akrydynowy. Mikroskop naświetla próbkę wzbudzeniem o krótkiej długości fali, takim jak światło ultrafioletowe lub niebieskie. Chromofory absorbują światło wzbudzenia i emitują światło widzialne o większej długości fali. Następnie światło wzbudzające jest filtrowane (częściowo dlatego, że światło ultrafioletowe jest szkodliwe dla oczu), tak że przez soczewkę oka przechodzi tylko światło widzialne, tworząc obraz preparatu w jasnych kolorach na ciemnym tle. Mikroskopy fluorescencyjne mogą identyfikować patogeny, znajdować określone gatunki w środowisku lub znajdować lokalizacje określonych cząsteczek i struktur w komórce. Opracowano także metody odróżniania komórek żywych od martwych na podstawie tego, czy pochłaniają one określone fluorochromy. Czasami na tej samej próbce stosuje się wiele fluorochromów, aby pokazać różne struktury lub cechy. Jednym z najważniejszych zastosowań mikroskopii fluorescencyjnej jest immunofluorescencja, która służy do identyfikacji niektórych drobnoustrojów poprzez obserwację, czy wiążą się z nimi przeciwciała. (Przeciwciała to cząsteczki białka wytwarzane przez układ odpornościowy, które przyłączają się do określonych patogenów, aby je zabić lub zahamować.) W tej technice stosuje się dwa podejścia: bezpośredni test immunofluorescencyjny (DFA) i pośredni test immunofluorescencyjny (IFA). W DFA specyficzne przeciwciała (np. te, które są skierowane przeciwko wirusowi wścieklizny) barwi się fluorochromem. Jeśli próbka zawiera docelowy patogen, pod mikroskopem fluorescencyjnym można obserwować przeciwciała wiążące się z patogenem. Jest to barwienie przeciwciałem pierwotnym, ponieważ wybarwione przeciwciała przyłączają się bezpośrednio do patogenu. W IFA przeciwciała wtórne barwi się fluorochromem, a nie przeciwciałami pierwotnymi. Przeciwciała wtórne nie przyłączają się bezpośrednio do organizmu, ale wiążą się z przeciwciałami pierwotnymi. Kiedy niezabarwione przeciwciała pierwotne wiążą się z patogenem, można zaobserwować fluorescencyjne przeciwciała wtórne wiążące się z przeciwciałami pierwotnymi. Zatem przeciwciała wtórne przyłączają się pośrednio do patogenu. Ponieważ do przeciwciała pierwotnego może często przyłączać się wiele przeciwciał wtórnych, IFA zwiększa liczbę przeciwciał fluorescencyjnych przyczepionych do próbki, ułatwiając wizualizację jej cech. Ten tekst jest zaadaptowany z Openstax, Mikrobiologia 2e, Sekcja 2.4: Barwienie preparatów mikroskopowych.

Mikroskopia immunofluorescencyjna

Mikroskop fluorescencyjny wykorzystuje fluorescencyjne chromofory zwane fluorochromami, które mogą pochłaniać energię ze źródła światła, a następnie emitować tę energię jako ...

Education

JoVE Core

Cell Biology

Biologia komórki

Unit 1

Jednostka 1

Visualizing Cells, Tissues, and Molecules

Wizualizacja komórek, tkanek i cząsteczek

KEY TERMS AND CONCEPTS

Kluczowe terminy i pojęcia

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

JoVE Core Cell Biology illustrates complex cellular processes in easy-to-understand yet comprehensive animated video lessons. This collection focuses on the essential components and functions of cells, including organelles and membranes, cellular transport, and cell energetics. It is complemented by scientist-in-action videos that demonstrate the application of cell biology in real-world research experiments.

Fluorescent Antibody Staining in Cells

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