Immunofluorescence

Preparation of Fluorescence

To make fluorochrome-labeled antibodies, a fluorochrome must be tagged or conjugated to the antibody. Likewise, an antigen can also be conjugated to the antibody with a fluorescent probe in a technique called fluorescent antigen technique. Staining procedures can apply to both fixed antigen in the cytoplasm or to cell surface antigens on live cells, called “membrane immunofluorescence.” It is also possible to label the complement of the antibody-antigen complex with a fluorescent probe. In addition to the element to which fluorescence probes are attached, there are two general classes of immunofluorescence techniques: primary and secondary. The following descriptions will focus primarily on these classes in terms of conjugated antibodies.

There are two classes of immunofluorescence techniques, primary (or direct) and secondary (or indirect).

Primary (direct)

Primary, or direct, immunofluorescence uses a single, primary antibody, chemically linked to a fluorophore. The primary antibody recognizes the target molecule (antigen) and binds to a specific region called the epitope. The attached fluorophore can be detected via fluorescent microscopy, which, depending on the messenger used, will emit a specific wavelength of light once excited. Direct Immunofluorescence, although somewhat less common, has notable advantages over the secondary (indirect) procedure. The direct attachment of the messenger to the antibody reduces the number of steps in the procedure, saving time and reducing non-specific background signal. This also limits the possibility of antibody cross-reactivity and possible mistakes throughout the process. However, since the number of fluorescent molecules that can be bound to the primary antibody is limited, direct immunofluorescence is substantially less sensitive than indirect immunofluorescence and may result in false negatives. Direct immunofluorescence also requires the use of much more primary antibody, which is extremely expensive, sometimes running up to $400.00/mL.

Secondary (indirect)

Secondary, or indirect, immunofluorescence uses two antibodies; the unlabeled first (primary) antibody specifically binds the target molecule, and the secondary antibody, which carries the fluorophore, recognises the primary antibody and binds to it. Multiple secondary antibodies can bind a single primary antibody. This provides signal amplification by increasing the number of fluorophore molecules per antigen. This protocol is more complex and time-consuming than the primary (or direct) protocol above, but allows more flexibility because a variety of different secondary antibodies and detection techniques can be used for a given primary antibody.

This protocol is possible because an antibody consists of two parts, a variable region (which recognizes the antigen) and constant region (which makes up the structure of the antibody molecule). It is important to realize that this division is artificial and in reality the antibody molecule is four polypeptide chains: two heavy chains and two light chains. A researcher can generate several primary antibodies that recognize various antigens (have different variable regions), but all share the same constant region. All these antibodies may therefore be recognized by a single secondary antibody. This saves the cost of modifying the primary antibodies to directly carry a fluorophore.

Different primary antibodies with different constant regions are typically generated by raising the antibody in different species. For example, a researcher might create primary antibodies in a goat that recognize several antigens, and then employ dye-coupled rabbit secondary antibodies that recognize the goat antibody constant region ("rabbit anti-goat" antibodies). The researcher may then create a second set of primary antibodies in a mouse that could be recognized by a separate "donkey anti-mouse" secondary antibody. This allows re-use of the difficult-to-make dye-coupled antibodies in multiple experiments.

Limitations

As with most fluorescence techniques, a significant problem with immunofluorescence is photobleaching. Loss of activity caused by photobleaching can be controlled by reducing or limitation the intensity or time-span of light exposure, by increasing the concentration of fluorophores, or by employing more robust fluorophores that are less prone to bleaching (e.g., Alexa Fluors, Seta Fluors, or DyLight Fluors). Some problems that may arise from this technique include autofluorescence, extraneous undesired specific fluorescence, and nonspecific fluorescence. Autofluorescence includes fluorescence emitted from the sample tissue or cell itself. Extraneous undesired specific fluorescence occurs when a targeted antigen is impure and contains antigenic contaminants. Nonspecific fluorescence involves the loss of a probe's specificity due to fluorophore, from improper fixation, or from a dried out specimen.

Immunofluorescence is only limited to fixed (i.e., dead) cells when structures within the cell are to be visualized because antibodies do not penetrate the cell membrane when reacting with fluorescent labels and antigenic material must be fixed firmly on the site of its natural localization inside the cell. Intact antibodies can also be too large to dye cancer cells in vivo. Their size results in slow tumor penetration and long circulating half-life. Research has been done investigating the use of diabodies to get around this limitation. Proteins in the supernatant or on the outside of the cell membrane can be bound by the antibodies; this allows for living cells to be stained. Depending on the fixative that is being used, proteins of interest might become cross-linked and this could result in either false positive or false negative signals due to non-specific binding.

An alternative approach is using recombinant proteins containing fluorescent protein domains, e.g., green fluorescent protein (GFP). Use of such "tagged" proteins allows determination of their localization in live cells. Even though this seems to be an elegant alternative to immunofluorescence, the cells have to be transfected or transduced with the GFP-tag, and as a consequence they become at least S1 or above organisms that require stricter security standards in a laboratory.

Advances in Immunofluorescence

Many improvements to this method lie in the improvement of fluorescent microscopes and fluorophores. Super-resolution methods generally refer to a microscope's ability to produce resolution below the Abbe limit (a limit placed on light due to its wavelength). Super-resolution in fluorescence, more specifically, refers to the ability of a microscope to prevent the simultaneous fluorescence of adjacent spectrally identical fluorophores. A main example of a super-resolution fluorescent microscope method is called Stimulated Emission Depletion, otherwise known as STED.