Immunofluorescence is a semiquantitative technique that utilizes fluorophores to visualize various cellular antigens. Immunofluorescence is often used in research to detect protein expression and visualize the localization of cellular components within cells, tissues, or other 3D culture-derived structures. Experimentation is simple and efficient; first the sample is incubated with an antibody specific to the protein of interest. The antibody may be coupled to a fluorophore or may be detected by a secondary antibody that was previously conjugated. Next, target proteins or antigens are visualized via microscopy. Immunofluorescence is more sensitive than counterpart immunodiffusion methods and offers much faster testing times.

Among other things, immunofluorescent techniques have readily been applied to autoimmune disease research, to define antigen-antibody interactions on a subcellular level, and to identify small cell surface structures. Outside of research, immunofluorescence also holds clinical importance. It has routinely been used to detect and measure antibodies associated with immune-mediated inflammatory diseases as well as antibodies to bacteria, viral, protozoal, or parasites.

Before starting any immunofluorescence procedure, correct sample targets must first be chosen either from cultured cells, cell suspensions, tissue samples, or in entire organisms or organoids (termed whole-mount immunofluorescence). Fresh samples can also be used if they are snap-frozen or placed in an appropriate transport medium. Next, samples are fixed to prevent autolysis, mitigate putrefaction, and preserve morphology while maintaining antigenicity.

 

Fixation serves to immobilize target antigens without disturbing cellular architecture while also allowing antibodies better access to the target sites. As there is no universal fixative for every antigen, reagents and methods should be empirically determined. Chemical fixatives may preserve the immunoreactivity of a particular epitope, while degrading or masking other epitopes.

Cross-linking reagents are often used as they have the ability to form intra- and intermolecular methylene connections. Organic solvents may alternatively be preferred since they remove lipids, dehydrate cells, denature and precipitate cellular components. Next, tissue samples are embedded into paraffin to solidify them for sectioning. This step allows for dyes, probes, and antibodies to reach target sites without obstruction. Paraffin sections are then cut and mounted onto glass slides, undergo deparaffinization, and are then rehydrated. Now, it is necessary to restore epitope-antibody reactivity through antigen retrieval. This step is largely dictated by the target antigen identity, antibody character, tissue type, the method and duration of fixation. Antigen retrieval may be necessary if it is possible that the reactivity of the sample will be altered during fixation.

Another critical step in any immunofluorescent technique is in choosing proper antibodies. The primary antibody should be derived from a different species than the sample. This prevents the secondary antibody from cross-reacting with endogenous immunoglobulins (IgG) in the sample, which limits potential background staining. The secondary antibody must also work against the host species of the primary antibody. Secondary antibodies can also be further modified for visualization and signal amplification purposes.

Commonly, secondary antibodies are conjugated with fluorescent labels that emit light upon excitation. Enzymatic labels can also be conjugated to secondary antibodies that react with chromogen substrates for colorimetric analyses. For greater signal amplification, biotinylated or polyclonal secondary antibodies can be used. Polyclonal antibodies work by recognizing multiple epitopes, which increases binding and signal levels. Alternatively, biotinylated antibodies can bind with multiple fluorochrome-protein complexes, like avidin or streptavidin, to also amplify signals.

Prior to antibody application, however, a blocking step should be applied to prevent non-target binding. Blocking reagents should be those that have little to no affinity for the target epitopes, provide high binding rates to non-target sites, all while not disrupting cell morphology. Protein solutions can be used as blocking buffers and will bind most of, if not all, proteins present in the sample. Normal serum can also be used, which contain antibodies from the same species, and are capable of blocking non-target reactive sites to which secondary antibodies would otherwise bind. Protein-free buffers, alternatively, are widely available with options specific for various antibodies. Common blocking buffers include bovine serum albumin (BSA), non-fat dry milk, and gelatin though in general, blocking reagents must be determined empirically.

For the most part, immunofluorescent techniques are performed in one of two methods; in direct or indirect methods. Though the direct method is quicker, the indirect method is more widely employed due to its higher sensitivity, increased signal amplification, and advantageous ability to detect several targets simultaneously. In detection, fluorophore-conjugated antibodies emit light upon excitation which can be fluorescently observed. The ideal fluorophore for a particular experiment should be empirically determined considering a number of factors, including whether a confocal versus an epifluorescence microscope will be the imaging platform. Many manufacturers include fluorophore reference charts indicating maximum excitation and emission (Ex/Em) wavelengths which may help guide a researcher to appropriate use.

Additionally, the extinction coefficient and quantum yields of a chosen fluorophore must be taken into account. Fluorescence within the sample may appear dim if the extinction coefficients and quantum yield of the fluorophores are poor. To minimize photobleaching, photostable fluorophores can be selected instead, excitation duration and intensity can be reduced, and an antifade mounting step may be added to the protocol. In some cases multiple fluorophores may be used in tandem where typically dimmer fluorophores detect abundant antigens while brighter fluorophores detect sparse antigens. This, too, must be empirically determined.

Indirect or secondary immunofluorescence is commonly used in clinical laboratories to screen for antibodies-antigen interactions. First a specimen is incubated with an unlabeled primary antibody which binds the target molecule. The sample is then washed and incubated, typically, with a fluorochrome- conjugated anti-IgG antibody. The purpose of this second antibody is to reveal the presence of the first. A single primary antibody can also be bound by multiple secondary antibodies, thereby providing an amplified signal due to the presence of more fluorophore molecules. Since primary and secondary antibodies are derived from different species, numerous possibilities for antibody-antibody structures exist. For example, primary antibodies from a rabbit can be combined with fluorescently-coupled goat secondary antibodies creating a goat-anti-rabbit combo.

Indirect immunofluorescence is more flexible than its direct counterpart, and this technique often produces a brighter fluorescence with higher signal amplification, typically 6 to 8 fold. Indirect immunofluorescence is also essential if the monoclonal antibody is only available in the form of a cultured supernatant. The indirect method does, however, take more time to complete. Additionally, the anti-igG reagent cannot easily distinguish between exogenous and endogenous IgGs. For example, the rabbit-anti-mouse IgG reagent will always detect IgG on mouse immune cells, regardless if other antibodies are present. For this reason, many commercial antisera to mouse IgG have been depleted of anti-human antibodies. Such cross-reactivity requires that controls must be tested alongside samples in which the first antibody is omitted.

Direct immunofluorescence involves the chemical linking of a single primary antibody to a fluorophore. In the process, the primary antibody will locate the epitope and bind tightly during incubation. All unbound antibodies are then removed through a series of washing steps. One advantage of this technique is because the messenger attaches directly to the antibody, there is less possibility of antibody cross-reactivity which provides less nonspecific background signals. There are also fewer steps in the procedure, comparatively, which makes it slightly faster and less error-prone. One downside to direct immunofluorescence is that the number of fluorescent molecules able to bind to a primary antibody is limited. Also, the technique is significantly less sensitive than its indirect counterpart, and false negatives may occur. Lastly, the direct methods require a significant amount of primary antibodies which can be expensive.

Other variations of the immunofluorescence also exist. The salt split technique and antigen mapping technique, for example, are both well suited for use in skin samples and have shown clinical promise in the detection of bullous diseases. In the double staining method, or multicolor immunofluorescence, the distribution of two or more different antigens can be examined in the same sample. Double staining can either be performed simultaneously using an antibody cocktail, or by probing antigens sequentially. Double staining follows similar steps as the indirect method though with considerably different blocking and incubation steps. In the sandwich technique fluorescence of the antigen is measured between two layers, the capture and detection antibodies. This means the target antigen must contain at least two antigenic sites. The sandwich technique is beneficial as monoclonal or polyclonal antibodies can both be used.

Lastly, the calcium enhancement indirect technique has shown a significant increase in the sensitivity of immunofluorescent assays by the use of calcium-supplemented buffers. Such findings may also benefit procedures designed to purify and/or detect particular antigens in particular samples.

Immunofluorescence offers inherent advantages over other techniques with regards to signal amplification, targeting specificity, resolution, and analytical capabilities. It is simple and reproducible, offers a short procedure time of 1-3 hrs, has high sensitivity, and can provide substantial prognostic value. Methods have the capacity for further improvement and optimization through the use of polyclonal antibodies in conjugation with enzyme complexes. Additionally, immunofluorescence allows for multiplexing, can be used in a high-throughput platform, and is ideal for co-localization studies.

Depending on the proper selection of fluorophores, multiple antigens can be stained simultaneously without the concern of spatial orientation since different fluorophores are only sensitive to their corresponding excitation wavelengths. Fluorescent detection also offers better image qualities and semi-quantitative results. Confocal fluorescence microscopes can obtain higher resolutions and multiplanar images, and avoid the issues of fuzzy images resulting from chromogenic enzyme precipitates.

Some limitations with immunofluorescence, however, lie in fluorescence overlap and nonspecificity. Fluorescence signals depend on the quality, concentration, and selection of antibodies as well as proper handling of the specimen. Photobleaching remains an issue and can reduce activity within a sample, which simultaneously reduces observable data. This issue can be mitigated by limiting overall light exposure, by increasing the ratio of fluorophores employed, or by using specialized fluorophores. Autofluorescence may also occur within a sample when an undesired extraneous fluorescence is emitted from the sample, when a targeted antigen is contaminated, when a fluorophore improperly fixates, or a specimen is too dry.

The quality and concentration of the labeled antibody must also be taken into consideration to achieve a high fluorescent yield. Too much nonspecific antibody binding may not allow accurate antigen or protein localization, while a diluted antibody may not give off a sufficient readable signal.

Another limitation in immunofluorescence is that the technique is limited to fixed cells because antibodies cannot penetrate the cell membrane when reacting with fluorescent labels. It has been shown, however, that binding proteins in the supernatant or on the exterior of a cell membrane has allowed living cells to be stained. To counteract this issue, recombinant proteins that contain fluorescent protein domains such as green fluorescent protein (GFP) may be used within living cells. It is also important to note that in immunofluorescence some target proteins might become cross-linked which could result in false negatives or false positives.

Immunofluorescence
An introduction to Performing Immunofluorescence Staining
Immunofluorescence - Types, Techniques, and Limitations
Salt split technique: a useful tool in the diagnosis of subepidermal bullous disorders
Double immunofluorescent staining of rat macrophages in formalin-fixed paraffin-embedded tissue using two monoclonal mouse antibodies
Calcium enhances the sensitivity of immunofluorescence for pemphigus antibodies
Immunofluorescence