Immunofluorescence (IF) is a powerful method for visualizing intracellular processes, conditions and structures. IF preparations can be analyzed by various microscopy techniques (e.g. CLSM, Epifluorescence, TIRF, GSDIM), depending on the application or the researcher’s interest. Meanwhile, IF has become indispensable for a large number of research groups which have at least access to a simple fluorescence microscope.   The centerpiece of an IF experiment is a combination of two different components:   ● First, specific antibodies, which are used to form an immune complex to mark the desired molecules – in most cases proteins – in the cell.   ● Second, fluorochromes, which are coupled to the immune complexes and therefore visualize the target structures during microscopy.

Fig. 1: This chart illustrates a typical workflow of indirect IF with epithelial cells adherently growing on coverslips. After cultivation, cells get fixed and therefore killed with a chemical crosslinker (e.g. formaldehyde). Next a permeabilization step with detergents is performed to enable the antibodies to cross the cellular membranes. Blocking with normal serum, milk powder or bovine serum albumin reduces the unspecific binding of antibodies to non-target structures in order to minimize false-positive signals. Next the incubation with the first antibody takes place, which specifically recognizes epitopes on the target molecule. In a second incubation step the fluorescence-coupled secondary antibody is applied which binds to the first antibody and therefore visualizes the target structure. After antibody incubation, nuclei staining is performed with dyes such as DAPI or Hoechst which intercalate into DNA. After mounting of the coverslip with a mounting medium (e.g. Mowiol® or Vectashield®) on a microscope slide, the IF preparation is ready for microscopy.

Depending on the type of experiment there are two different IF variants: In direct or primary IF a specific primary antibody which is linked to a fluorochrome is used for binding to the target structure and its direct visualization.

In the second variant, referred to as indirect or secondary IF, a two-step incubation is performed. Firstly, a specific primary antibody recognizes the target structure. Then a fluorochrome-coupled secondary antibody is applied which specifically binds to the primary antibody. This specificity is obtained by directing the secondary antibody against the species in which the primary antibody was raised (see Antibodies and fluorochromes). Comparing the two IF variants, each of them has different advantages and drawbacks:

By coupling the primary antibody with a fluorochrome, direct IF is faster than the indirect version as time-consuming washing and incubation steps are omitted. Thus, direct IF is easier to handle and therefore suitable for the rapid analysis of samples in standardized IF experiments, for example in clinical practice. However, it is essential to employ a well-functioning primary antibody with high affinity to its antigen. This is simultaneously a negative aspect, as fluorescence-coupled and validated primary antibodies are expensive. Moreover, you need a separate primary antibody for each target structure, and the linkage of the antibody with a fluorochrome in direct IF restricts your flexibility in designing your experiment in comparison to indirect IF.

This flexibility is a significant advantage of indirect IF. Usually, several different target structures in the same specimen need to be visualized during one IF reaction, therefore a discrete fluorochrome must be selected for each target molecule. In indirect IF, different fluorescence-coupled secondary antibodies can be combined with different primary antibodies (considering the species reactivity, of course). In contrast, if you want to "play" with color combinations of your target structures in direct IF, you need an individual primary antibody for each color. Another advantage of the indirect method is signal amplification by the secondary antibody. Multiple secondary antibody molecules can bind to one primary antibody, which leads to an increase of fluorescence, meaning that less primary antibody has to be applied.

The workflow of indirect IF may take more time, but due to the possible combinations of primary and secondary antibodies and a generally more economical procedure it is the method of choice for most researchers.

Fig. 2: There are two ways to visualize a target structure by immunofluorescence: In both variants a specific first antibody is used to recognize a particular epitope on the target molecule. Here, the target molecule consists of several identical protein subunits (= macro molecule) and therefore it exhibits several epitopes of the same type on each subunit. For simplification only one epitope is depicted here.
In direct IF the first antibody is directly linked to a fluorochrome which visualizes the target structure under the microscope.
In indirect IF a fluorescence-coupled secondary antibody is applied during a second incubation step which specifically labels the first antibody. This leads to a greater flexibility in choosing antibodies and fluorochromes and furthermore to a signal amplification, because several secondary antibody molecules can bind to one primary antibody.

The most crucial tool for an IF staining of high quality is a good primary antibody. Meanwhile, one or even several commercially purchasable antibodies are available for approximately each protein in almost every cell type. However, there are a few extremely important things to note here.

To make a precise scientific or clinical statement based on your IF staining you must ensure the specificity of your primary antibody to its target antigen. In doing so you should not exclusively rely on the statements of your commercial providers. Choose your antibody according to already used and validated primary antibodies in literature on the subject. Check the antibody’s datasheet on the manufacturer’s website for available pictures of IF stainings and compare them with your expectations or other published illustrations. Take notice of the antibody’s clonality, as monoclonal antibodies specifically bind to only one epitope, whereas polyclonal antibodies recognize several epitopes, making non-specific labeling of non-target structures more likely. Therefore, monoclonal antibodies with their high specificity and good affinity are usually more expensive, but they also achieve better results.

If you want to perform multicolor indirect IF, the different primary antibodies must derive from different species in order to distinguish the immune complexes by labeling with fluorescence-coupled secondary antibodies afterwards (see Table 1). Say, for example, you are performing IF with antibody anti-protein A derived from mouse, antibody anti-protein B from rabbit and antibody anti-protein C from rat. When choosing the secondary antibodies you have to remember that each of them specifically recognizes only one primary antibody. Furthermore, the fluorochromes in this example of the three secondary antibodies must differ in their wavelength spectrum to discriminate their fluorescence signal in microscopic analysis. Nowadays, secondary antibodies linked to fluorochromes with a wavelength range from ultraviolet to infrared against primary antibodies from almost any species are purchasable. Thus the researcher is only limited by the configuration of the available microscopes (filter sets, excitation lasers).

Tab. 1: This example of a multicolor indirect IF demonstrates how to label simultanously three different proteins in the same cell. The three first antibodies must derive from different species in order to detect them with three different fluorochrome-coupled secondary antibodies. The secondary antibodies’ fluorochromes must differ in their wavelenght spectrum for a distinct analysis in the microscope. The cell depicted in the sample image was also treated with Hoechst 33342 for nucleus staining. Microscopy was performed on a Leica TCS SP2.

IF protocols exist for a variety of different specimens or samples. The simplest and most commonly used method is the staining of cultured (eukaryotic) cells from cell culture. Adherently growing cells can be seeded on coverslips, multiwell-inserts or directly on glass bottom culture dishes and used for IF at the desired time. IF is also possible with suspension cells after application of the cells to an object slide, e.g. by cytospin. In both cases, the focus is on the analysis of intracellular processes or structures, and this is designated immunocytochemistry (ICC).

In immunohistochemistry (IHC), on the other hand, the presence of proteins or molecules is examined in a tissue-specific context. Here, ultrathin sections of organ preparations (usually embedded in paraffin wax) are used to e.g. investigate the expression of proteins in a healthy organ compared to a diseased one. In addition to the preparation of tissue sections it is also possible to perform IF with whole organisms, a process referred to as "whole mount IHC". For this purpose, embryos of different model organisms such as mouse, chicken or zebrafish, for instance, or plant model organisms like Arabidopsisthaliana are used. In whole mount IHC one is limited by the size of the specimen and the associated penetration depth of the IF reagents. The separate incubation steps take longer than the staining of cultured cells. Also, microscopes with special optical equipment for the analysis of large specimens have to be available.

Fig. 3: a) The Immunhistochemistry (IHC) staining of a human kidney displays different cell types in different structures (e.g. Glomerolus, proximal tubule, distal tubule). The expression of the protein labeled with a green fluorochrome is limited to a specific cell type, whereas the red-labeled protein is widely expressed.
b) The Immuncytochemistry (ICC) image shows staining of two proteins in cells of the same type (MDCK) by indirect IF. Here, a tissue-specific study is not possible, but the analysis if both proteins colocalize in the same structure. Both samples were treated with Hoechst 33342 for nucleus staining. Microscopy was performed on a Leica TCS SP2.