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Labeled antibodies allow you to target almost any cellular target in a fixed and permeabilized cell

Because of their specificity and the relative ease with which they can be obtained, antibodies represent an essential toolset for biology researchers.

Learn the pros and cons for direct and indirect immunofluorescence labeling methods and see the basic considerations for choosing a primary antibody for your experiment.


What is immunofluorescence?

Immunofluorescence is a technique for fluorescently labeling a specific biological target within a sample using an antibody. An antibody is a Y-shaped high–molecular weight glycoprotein, also called an immunoglobulin, that binds specifically (but noncovalently) to another molecule (often called the antigen or epitope). In immunofluorescence, the specificity of the fluorescent label comes from the specificity of the antibody for its antigen; the detection of the bound antibody is due to the fluorophore that is attached to the antibody.

You’ll also see immunofluorescence referred to as both immunocytochemistry (ICC) and immunohistochemistry (IHC), and sometimes even antibody labeling. IHC generally refers to experiments where targets within thin sections of tissue are stained, while ICC refers to staining cells that have been isolated from tissue by removing the extracellular matrix they originally resided in, or staining cultured cells.

Figure 1. Immunofluorescence techniques rely on a a fluorophore-conjugated antibody, which has specificity for a specific target. The illustration shows an antibody (grey) conjugated to a fluorophore (green). In reality, most commercially conjugated antibodies would be labeled with 2–7 of the same fluorophore molecules per antibody.

  Drawing showing a Y-shaped antibody molecule with a fluorophore bound to the Fc region


Both primary and secondary antibodies are used in immunofluorescence

When antibodies are discussed in the context of fluorescence imaging, you will hear the terms primary or secondary antibody. These terms refer to the order in which the fluorescently labeled antibody binds the antigen of interest. Primary antibodies bind directly to the target, while secondary antibodies bind indirectly by using the primary antibody as a bridge to the targeted biomolecule.

Both the host species and the antigen species are used to name antibodies

When you are selecting antibodies, you will see terms like goat anti-mouse, or donkey anti-goat, and it can be a little hard to keep these straight. The first animal mentioned in these terms refers to the host species, i.e., the species in which  the antibody was raised. The animal name that follows the “anti” tells you what species the antibody recognizes. To produce secondary antibodies for immunolabeling, the antigen will always be the antibody of another species. So a goat anti-mouse IgG Alexa Fluor® 488 is a goat antibody that was raised against mouse immunoglobulins and is conjugated to a dye that emits at 488 nm.


Comparison of direct and indirect immunofluorescence methods

Both direct and indirect methods of immunofluorescence have advantages and disadvantages. What method you choose to use for your immunofluorescence imaging depends a lot on your target and what primary antibodies are available. In general, using fluorescent dye–conjugated secondary antibodies in an indirect labeling approach is the most common and cost effective, since fluorescently labeled secondary antibodies are relatively inexpensive, come in a wide array of colors, can be used in conjunction with any primary antibody that they are reactive to, and allow amplification of your signal, since more than one secondary antibody can bind to the primary antibody.

Diagram showing a direct immunofluorescence scenario with fluorophores bound to the antibody, compared to to an indirect immunofluorescence scenario where labeled antibodies are bound to the antibody

Figure 2. Immunolabeling can be accomplished in several ways. The antibody which binds a specific target or epitope is shown in orange. For direct immunofluorescence, the antibody binding the epitope is labeled with fluorophores (green). For indirect or secondary detection, the primary antibody binds the epitope and a fluorophore-labeled secondary antibody (purple) that has specificity for the primary antibody binds to it.


Direct immunofluorescence

  • Direct labeling results in shorter sample staining time and a simpler workflow
  • Offers the best solution for specific targeting if multiple antibodies to multiple targets are raised in the same species
  • Avoids any cross-reactivity between secondary antibodies
 
  • Fluorophore signal relies on the finite number of fluorophores that can be attached to a single antibody, and consequently, can limit detection to high-abundance targets
  • Workflow can be less flexible (fewer colors may be available), more expensive, and, if commercially labeled direct conjugates are unavailable, more difficult


Indirect immunofluorescence or secondary detection

  • Offers greater sensitivity because more than one secondary antibody can bind to each primary antibody, resulting in signal amplification
  • Commercially produced secondary antibodies are relatively inexpensive, and are available in a wide spectrum of colors
 
  • Potential for cross-reactivity; when performing multilabel experiments, indirect labeling requires the use of primary antibodies that are not raised in the same species
  • Secondary antibody may react with endogenous immunoglobulin in tissue samples, which can result in higher background fluorescence


Selecting a primary antibody

One of the most important things to consider in planning your immunofluorescence experiment is the selection of your primary antibody, as it is the main cause of success or failure. The best way to pick from commercially available primary antibodies is to choose the one that you know works for immunofluorescence techniques. You can usually find examples of use in the description of the antibody or in published literature. If you can, you will want to select a primary antibody that was raised against the same species as the target in your sample. However, this isn’t always possible and it doesn’t mean your experiment is doomed. It just means you need to see what other species have proteins with high amino acid homology to the sequence of your target, and pick a primary antibody raised against the most homologous target.

When you are in the process of selecting your antibodies, it’s a good idea to consider ahead of time what other targets you may wish to image in the same experiment. If you are going to use antibodies for more than one color of fluorescence, you will want to be sure to avoid having more than one primary antibody raised in the same host species. For example, if you have one unconjugated primary antibody whose host animal was mouse, you will want to make sure that your conjugated secondary antibody was raised against mouse antibodies in a host that is not mouse. If you have a second target, you will need to ensure that the host species for that primary antibody is something other than mouse, so that your additional conjugated secondary antibody reacts only with one primary (and not both).

Schematic diagram showing how primary antibodies raised in different host species are only bound by secondary antibodies specific for that host species, which helps prevent cross-reactivity.

Figure 3. Secondary detection of two different targets in the same sample relies on the primary antibody for each target being raised in different host species to avoid cross-reactivity between the secondary antibodies.


Selecting a secondary antibody

Once you have the primary antibodies sorted out, choosing the commercially available secondary antibody is a lot easier. You want to choose a secondary antibody that was raised against the same species as the host of the primary antibody you selected. If you have a primary antibody raised in mouse, you need to select a host species other than mouse for your secondary; for example, a goat anti-mouse conjugated to a fluorescent dye in the color you want to image in.

Troubleshooting

Sometimes a standard immunofluorescence assay using conjugated secondary antibodies still does not provide sufficient signal. This could be because your primary antibody is not very good, and by not very good, we mean either low affinity or not very specific for the target, or that the target is present in such low abundance in your sample that the amplification of signal provided by the conjugated secondary antibody is still too dim to be visualized easily. In either case, there are amplification strategies that could increase your signal.

Biotin-streptavidin amplification is one strategy, and results in an increase in the number of fluorophores that label your target. The disadvantage is that there can also be additional steps required to block endogenous biotin to prevent nonspecific labeling.

 

Drawing showing how multiple fluorescent streptavidin molecules can bind to a single antibody and boost the fluorescent signal

Figure 4. Biotinylated antibodies labeled using fluorophore-streptavidin conjugates can boost dim signals in your labeling experiments because more fluorophores can bind to each antibody molecule. Specificity for the epitope is retained.

Another strategy is to use tyrosine signal amplification, where the secondary is conjugated to an enzyme that can release reactive dyes that will label the immediate area surrounding the site of antibody binding in the presence of H2O2. Similarly to biotin-streptavidin amplification, tyrosine signal amplification adds additional steps to the staining workflow and may require additional blocking to rid your sample of unwanted high background fluorescence.

Drawing showing how enzyme-driven tyrosine signal amplification can boost the signal from a bound antibody

Figure 5. Tyrosine signal amplification can also amplify dim signals by adding many more fluorophores per epitope, but the labeling may be less specific due to reactive fluorophores binding to the immediate area surrounding the epitope.

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