IMMUNOFLUORESCENCE: RATIONALE, PROTOCOL, AND APPLICATIONS - BIOLOGY - 2024

The immunofluorescence is a powerful technique immunostaining using antibodies covalently bonded to fluorescent molecules to identify specific targets in cell samples fixed onto a solid support.

This technique involves microscopic observation with immunological specificity, making it possible to observe live or dead cells that can present minuscule amounts of antigens. It is widely used both in the field of research and in the clinical diagnosis of various pathologies.

Immunolabeling of actin filaments in cardiomyocyte cells (Source: Ps1415 via Wikimedia Commons)

This technique, mainly qualitative (with some quantitative variants), has to do specifically with the visualization of a sample by the product signal of a fluorophore, which is a fluorescent molecule bound to an antibody and which is capable of being excited at a certain wavelength.

In the cellular context it is very useful to study the presence / absence and subcellular location of proteins. The technique was initially used in the clinical setting for the diagnosis of viruses such as influenza and subsequently for many other infectious diseases.

It is a highly sensitive technique, and with the appropriate microscopy equipment, it can have very good resolution. It requires, for its observation, the use of confocal or epifluorescence microscopes.

However, despite being very popular, it can present some important problems with respect to obtaining non-specific fluorescence that generates some background “noise”, which often limits the adequate reading of the results.

Basis

Immunofluorescence is based on the exploitation of the biological phenomenon of the interaction reaction between an antibody and an antigen. It has to do specifically with the visualization or detection of this reaction by exciting fluorescent molecules to a specific wavelength.

An antibody is an immunoglobulin protein secreted from active B cells, which is specifically generated against an antigen, to which it can bind with high affinity and specificity. Immunofluorescence makes use of IgG immunoglobulins, which are found soluble in blood serum.

Antibodies are molecules up to 950 kDa made up of two short (light) and two long “Y” -shaped (heavy) peptide chains. Both the light and heavy chains are divided into two domains: one variable, capable of recognizing the antigen, and the other constant or conserved, characteristic of each species.

Antigens are functionally defined as molecules that can be recognized by an antibody and are, for the most part, proteins. When an animal is exposed to an antigen, the lymphocytes of the immune system are activated, producing specific antibodies against it and that function as a defense system.

An antigen, such as a protein, for example, may have more than one epitope or site of recognition by an antibody, thus the serum of the animal exposed to an antigen may have polyclonal antibodies against different regions of the same protein.

Immunofluorescence, then, exploits the ability of an animal to produce polyclonal antibodies against a specific antigen in order to purify it and subsequently use it for the detection of the same antigen in other contexts.

Among the fluorescent dyes or molecules most used for some immunofluorescence techniques are fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate-5 and 6 (TRITC), many cyanines such as Cy2, Cy3, Cy5 and Cy7 and dyes called Alexa Fluor®, such as the Alexa Fluor®448.

Protocol

The immunofluorescence protocol varies depending on many factors, however, in general terms, it encompasses a linear sequence of steps consisting of:

• Preparation of the plates and cells
• Fixation of samples
• Permeabilization
• Blocking
• Immunostaining or immunostaining
• Assembly and observation

-Preparation

Of the samples

The preparation of the samples will depend on their nature and the type of experience to be carried out. The simplest case, which involves the use of cells in suspension, will be explained below.

Cells in suspension, that is, in a liquid culture medium, must first be separated from it by centrifugation and then must be washed with a buffer solution or isosmotic "buffer", which preserves their integrity.

Normally, a phosphate-saline buffer known as PBS is used, in which the cells are resuspended and this mixture is centrifuged again to obtain the cells free of the culture medium, which may contain interfering substances.

Of the blades

The slides used for microscopic observation, where the cells will later be fixed for the corresponding downstream treatments, must also be carefully prepared.

These are covered or "sensitized" with a solution of poly-lysine, a synthetic polymer that will act as a "molecular glue" between the cells and the solid support, thanks to the electrostatic interaction between the positive charges of their amino groups and the cells. negative charges on the proteins that coat cells.

Fixation of samples

This process consists of immobilizing the proteins found inside the cell in order to keep their spatial location intact. The molecules used must be capable of crossing all types of cell membranes and forming lattices with the covalent proteins.

Formaldehyde and paraformaldehyde, glutaraldehyde and even methanol are widely used, with which cell samples are incubated for a certain time and then washed with an isosmotic buffer solution.

After fixing the cells, they continue to be attached to the sheets previously sensitized with poly-lysine.

Permeabilization

Depending on the type of test that is carried out, it will be necessary to permeabilize the cells under study or not. If what is sought is to know the location, presence or absence of a certain protein on the cell surface, permeabilization will not be necessary.

On the other hand, if you want to know the location of a protein inside the cell, permeabilization is essential and will consist of incubating the samples with Triton X-100, a detergent capable of permeabilizing cell membranes.

Blocking

A fundamental step in all immunological techniques is blocking. At this stage of the procedure, blocking consists of covering, on the sensitized sheets, all the sites with poly-lysine molecules to which cells did not adhere. That is, it prevents any nonspecific union.

Normally solutions with bovine serum albumin (BSA) in PBS buffer are used for blocking and the best results are obtained the longer the incubation time with this solution. After each step, including blocking, it is necessary to wash off the remaining solution.

Immunostaining or immunostaining

The immunostaining or immunostaining procedure will depend mainly on whether it is a direct or indirect immunofluorescence (see below).

If it is a primary or direct immunofluorescence, the samples will be incubated with the desired antibodies, which must be coupled to the fluorescent dyes. The incubation procedure consists of making a dilution of the antibody in a solution that will also contain BSA but in a lower proportion.

When the case is that of a secondary or indirect immunofluorescence, two consecutive incubations should be carried out. First with the desired antibodies and then with the antibodies that are capable of detecting the constant regions of the primary immunoglobulins. It is these secondary antibodies that are covalently bound to fluorophores.

The technique is very versatile, allowing simultaneous labeling of more than one antigen per sample, as long as there are primary antibodies coupled to different fluorophores, in the case of direct immunofluorescence.

For simultaneous labeling in indirect immunofluorescence, it is necessary to ensure that each primary antibody is produced in a different animal, as well as that each secondary antibody is coupled to a different fluorophore.

Like blocking, incubation with antibodies gives better results the longer the time of this. After each step, it is necessary to wash off the excess antibodies that did not bind to the samples and in the secondary immunofluorescence it is necessary to block before adding the secondary antibody.

Certain techniques use other stains that are not related to immunostaining, such as staining of nuclear DNA with the DAPI fluorophore.

Assembly and observation

During the final incubation time with the fluorophores it is necessary that the samples remain in the dark. For observation under the microscope, it is common to use some substances to preserve the fluorescence of the fluorophores coupled to the antibodies.

Types

Graphic summary of direct and indirect immunofluorescence (Source: Westhayl618 via Wikimedia Commons)

Direct or primary immunofluorescence

It has to do with the detection of antigens through the use of fluorescent antibodies. The main advantage of using this technique is its speed, however, many cases of nonspecific binding can occur in the process, particularly when studying human sera, as they are rich in highly heterogeneous antibodies.

Indirect or secondary immunofluorescence

It is also known as the "sandwich" technique and this involves the development of the technique in two steps. The first has to do with the use of a non-fluorescent antibody and its binding to the antigen of interest.

Against the constant region of this first antibody (which will now serve as antigen) a second antibody capable of recognizing it is used, which is associated with a fluorescent molecule.

The appearance of a fluorescent signal will be the result of specific recognition between the first non-fluorescent antibody and the antigen of interest; the presence of this first antibody conditions that of the second, which is labeled and thanks to which the presence or absence of the antigen can be determined.

Despite being a much more time-consuming technique than direct immunofluorescence (as it includes one more incubation step), this technique does not involve the design of a fluorescent antibody for each antigen that is studied, which results, in economic terms, more viable.

Furthermore, it is a more sensitive technique in terms of signal amplification, since more than one secondary antibody can bind to the constant region of the primary antibody, thus amplifying the intensity of the fluorescent signal.

Applications

As may have been noted previously, immunofluorescence is an extremely versatile technique, which has been used in many ways in the scientific and clinical fields. It can be used to answer ecological, genetic, and physiological questions regarding many organisms.

Among the clinical applications, it is used for the direct diagnosis of some dermatological diseases, either using direct or indirect immunofluorescence on epithelial tissue of the patients studied.

Immunofluorescence techniques have been available in unicellular organisms such as yeast to visualize intranuclear and cytoplasmic microtubules, actin and associated proteins, 10nm filaments, and other constituents of the cytoplasm, membrane, and cell walls.

References

1. Abcam, Immunocytochemistry and immunofluorescence protocol. Retrieved from abcam.com
2. Greph, C. (2012). Fluorescent Dyes. Retrieved from leica-microsystems.com
3. Miller, DM, & Shakest, DC (1995). Immunofluorescence Microscopy. In Methods in Cell Biology (Vol. 48, pp. 365–394). Academic Press, Inc.
4. Odell, ID, & Cook, D. (2013). Immunofluorescence Techniques. Journal of Investigative Dermatology, 133, 1–4.
5. Princle, BJR, Adams, AEM, Druain, DG, & Brian, K. (1991). Immunofluorescence methods for yeast. In Methods of Enzymology (Vol. 194, pp. 565–602). Academic Press, Inc.
6. Schaeffer, M., Orsi, E. V, & Widelock, D. (1964). Applications of immunofluorescence in Public Health Virology. Bacteriological Reviews, 28 (4), 402–408.
7. Vrieling, EG, & Anderson, DM (1996). Immunofluorescence in phytoplankton research: applications and potential. J: Phycol., 32, 1–16.