Immunofluorescence - Types, Techniques, and Limitations

What is immunofluorescence?

Immunofluorescence is one of the techniques used within light microscopy, especially on microbiological samples. It uses a fluorescence microscope to observe antibodies, bonded to their antigens, with fluorescent dyes attached to specific biomolecule targets. Observers can therefore visualize the distribution of target molecules throughout a sample. The specific antigen region that an antibody recognizes is called an epitope. Since many antibodies can attach to the same epitope, and there is variance between the levels of binding between antibodies and the epitopes they can recognize, efforts are being made to further develop and improve epitope mapping. The binding of a fluorophore to an antibody also cannot interfere with the immunological specificity of the antibody or with the binding capacity of its antigen.

Using antibodies to stain proteins is called “immunostaining,” and immunofluorescence is a commonly cited example of such a technique. Taking advantage of the antibody-antigen relationship in tissues has led to a field called immunohistochemistry. In this field, practitioners use fluorophores to visualize the location of the antibodies.

Immunofluorescence can be used to analyze the distribution of proteins, glycans and small molecules, both biological and non-biological. It can be used to visualize a wide range of samples, from intermediate-sized filaments to tissue sections, individual cells, or cultured cell lines.

Epitope insertion can be used with immunofluorescence to determine structures, in cases where the topology of a cell membrane is as yet undetermined.

Another use of immunofluorescence is as a semi-quantitative method of gaining insight into levels and localization patterns of DNA methylation. There is some subjectivity in the analysis of the methylation levels, and the method is more time consuming than true quantitative methods.

Immunofluorescence can also be used in fluorescence staining in combination with some non-antibody methods, such as DAPI in the labelling of DNA.

The simplest microscope design used for examples of immunofluorescence samples is the epifluorescence microscope. Another common design is the confocal microscope.
Also commonly used are various designs of super-resolution microscopes which are capable of much higher resolution levels.


Fluorochrome-labelled antibodies are made by conjugating a fluorochrome to the antibody, a process also known as tagging. A process called fluorescent antigen technique can also be used. In this process, an antigen is conjugated to the antibody with a fluorescent probe. Fixed antigen in the cytoplasm and cell surface antigens on living cells can both be stained via such procedures, which is known as “membrane immunofluorescence.” The complement of the antibody-antigen complex can also be labelled, or tagged, with a fluorescent probe.

Types of Immunofluorescence

Immunofluorescence techniques fall into two general classes: Primary, also known as direct; and secondary, also called indirect.

1. Primary (direct)

Primary immunofluorescence is a process which includes chemically linking a single, primary antibody to a fluorophore.

The primary antibody binds to a specific region of the target molecule. The region is called the epitope. The immune response system of an organism with adaptive immunity is thereby manipulated. Fluorescent microscopy can detect the attached fluorophore. The fluorophore, depending on the messenger used, is excited and emits a specific wavelength of light.

This technique is a little less common than the secondary, or indirect technique, but nonetheless has some advantages. Because the messenger attaches directly to the antibody (hence the alternate name “direct”), there is less possibility of antibody cross-reactivity, there are fewer steps in the procedure, which makes it faster and less likely to contain errors, and there is less non-specific background signal.

There are downsides too though. The number of fluorescent molecules that are able to bind to a primary antibody is limited. This makes the direct method significantly less sensitive than its indirect counterpart, and false negatives may occur. There is also the cost factor. Primary antibody is expensive, sometimes up to $400.00/mL, and the direct method uses a lot more of it than the indirect method does.

2. Secondary (indirect)

Secondary immunofluorescence, also called indirect immunofluorescence, uses two antibodies.

The first one (primary) is not labelled, and specifically binds the target molecule. The secondary antibody is the one that carries the fluorophore.

It recognizes the primary antibody and binds to it. A single primary antibody can be bound by multiple secondary antibodies, thereby providing an amplified signal because there are more fluorophore molecules per antigen. This process entails more steps than primary/direct methods and therefore takes more time. It is more flexible, however, as different secondary antibodies can be used, and more detection techniques are applicable, for a single type of primary antibody.

This process is based on the idea that we can divide each antibody into two theoretical parts, a variable region and a constant region. The first recognizes the antigen, and the second makes up the structure of the antibody molecule. The molecule is, in reality, composed of four polypeptide chains. Two of these are heavy chains, and two are light. Several primary antibodies can have different variable regions – meaning that they can recognize various antigens – while all sharing the same constant region. A single secondary antibody can therefore recognize all of them, so there is no need for the trouble or cost of modifying the primary antibodies to directly carry a fluorophore.

Raising the antibody in different species produces different primary antibodies (thought this is not the only method, it is the usual one). Primary antibodies from a goat might be combined with dye-coupled rabbit secondary antibodies that recognize the constant region of the goat antibody, creating “rabbit -anti-goat” antibodies. A second set might then be made from primary antibodies in a mouse that could be recognized by a separate “donkey anti-mouse” secondary antibody. Since the dye-coupled antibodies are difficult to make, and this process allows reuse of them in multiple experiments, the cost is reduced over several experiments.

Limitations of Immunofluorescence

Photobleaching can be a significant problem in immunofluorescence microscopy. Photobleaching can reduce the activity in a sample, simultaneously reducing observable data. It can be mitigated by limiting the overall light exposure, either by reducing intensity, or time span; by increasing the ratio of fluorophores employed; or by using fluorophores that are known to be less prone to bleaching. Examples of more robust fluorophores include Alexa Flours, Seta Fluors, and DyLight Fluors.

Autofluorescence can also be a problem. Undesired extraneous fluorescence (both specific and nonspecific) can be emitted from the sample tissue or cell, when a targeted antigen contains antigenic contaminants (specific fluorescence), or when a fluorophore improperly fixates, or a specimen is dried out, causing a loss in the probe’s specificity (nonspecific fluorescence).

Immunofluorescence is limited to fixed, or “dead” cells when the goal is to observe structures within the cell. This is because the antibodies do not penetrate the cell membrane when reacting with fluorescent labels. The site of natural localization inside the cell must be accessible for the antigenic material to properly affix to it. Cancer cells are often too small for some intact antibodies to dye them in vivo. This results in slow tumor penetration and a longer circulating half-life, though some research in the area of diabody use may help us to get around these problems. Binding proteins in the supernatant, or on the exterior of a cell membrane, can allow even living cells to be stained. Some proteins of interest might become cross-linked, though, depending on the fixative employed, and non-specific binding could result, giving false negative or false positive signals.

Another way to go about this is to use recombinant proteins that contain fluorescent protein domains such as green fluorescent protein, or GFP. These tagged proteins show their localization within live cells. Though this technique solves some scientific issues, it triggers others. Once the cells are transfected or transduced with the GFP tag, they become at least S1 or above organisms, and therefore require stricter security standards in the lab. The technique alters the genetic information of the cells.

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