The Fundamentals of Immunofluorescence Staining – A Brief Guide

Immunofluorescence staining is a routine yet powerful technique that allows researchers to detect the levels and locations of proteins in complex cellular and tissue architectures, providing unprecedented insights into human health and disease. The method is largely based on antigen-antibody binding followed by fluorescent detection, but many different types of immunofluorescence staining and antibodies are available depending on the experimental goal and needs of the researcher.

So, to help you understand direct from indirect immunofluorescence and primary antibodies from secondary antibodies, we’ve compiled a handy guide including the must-know information to nail your next immunofluorescence experiment with the most appropriate reagents possible.

What is Immunofluorescence Staining?

Immunofluorescence staining harnesses the inherent immunological properties of antibodies to recognize specific antigen targets, from proteins to small biological and non-biological molecules. By coupling these antibodies to fluorescent labels known as fluorophores, researchers can use microscopes to visualize where proteins reside in cells or whole tissues to provide an intricate, detailed snapshot of cellular structures, processes, and interactions, often with clinical implications.

Immunofluorescence staining has advanced clinical research since as early as 1941, when Albert Hewett Coons and colleagues reported the first use of fluorescently labeled antibodies to visualize pneumococcal antigens in infected tissue specimens (Coons, Creech and Jones, 1941).

This monumental achievement firmly opened the door to the advanced world of immunofluorescence that we know today, where simultaneous subcellular detection of multiple proteins is routine.

Despite the striking advances in antibody specificity, fluorophore labels, and microscope sensitivity over the past 70 years, the underlying principle developed by Coons remains largely unchanged. It can be broken down into two main categories depending on the research question and the type of antibodies used.

Direct or Indirect Immunofluorescence: What’s the difference?

The two core variants of immunofluorescence staining are direct immunofluorescence and indirect immunofluorescence.

In direct immunofluorescence, the primary antibody is already conjugated to a fluorophore to allow binding to the target antigen and its direct visualization by fluorescence microscopy in a one-step process.

In contrast, indirect immunofluorescence is a two-step sequential process where a primary antibody first recognizes its target. Then a fluorophore-coupled secondary antibody is applied to specifically bind to the primary antibody.

Both approaches have advantages and disadvantages that you must consider before commencing an immunofluorescence staining experiment.

Direct Immunofluorescence: Simple and Fast

Direct immunofluorescence is more straightforward and faster than indirect immunofluorescence, as the primary antibody is already coupled to the fluorophore, removing time-consuming washing and incubation steps. Therefore, direct immunofluorescence lends itself to rapid analysis of samples or automation in clinical settings. Also, background fluorescence is often reduced due to less non-specific binding and minimal species cross-reactivity.

For direct immunofluorescence, if an experiment aims to visualize multiple proteins simultaneously for cell-type identification or their co-localization, finding appropriate reagents for multiplex experiments can be challenging as each primary antibody must be conjugated to a different fluorophore, which for less popular antibodies might not always be commercially available.

To make multicolor direct immunofluorescence experiments more accessible to researchers, Antibodies Inc. now offers over 400 primary antibodies conjugated to 490 nm, 550 nm, 594 nm, or 650 nm fluorophores to provide users with maximum flexibility, even for multiplexed experiments. This includes our highly cited anti-tyrosine hydroxylase antibody, now available conjugated to four different fluorophores.

Indirect Immunofluorescence: Sensitive and Flexible

Despite its advantages, direct immunofluorescence can sometimes be less sensitive than indirect immunofluorescence, since in indirect immunofluorescence, several secondary antibody molecules bind to one primary antibody, resulting in a stronger signal.

Similarly, indirect immunofluorescence is often a more flexible option than direct immunofluorescence, thanks to the vast number of unconjugated primary antibodies and fluorophore-coupled secondary antibodies available to label diverse targets with specific colors instead of relying only on fluorophore-coupled primary antibodies.

For instance, we provide over 2000 unconjugated primary antibodies with reactivity in many different species for indirect immunofluorescence. We are consistently adding to our collection with new products like our recent brain-validated SARM1 and CSPG4/NG2 monoclonal antibodies, also available as fluorophore-conjugates. We also provide over 30+ different secondary antibody options for ultimate flexibility in your immunofluorescence experiments.

Thanks to the flexibility of indirect immunofluorescence, it has become ubiquitous in biological research. However, as with any technique, the results gained are only as good as the reagents and the experimental design. Therefore, it is crucial to use the best primary and secondary antibodies available to provide rich and accurate molecular maps pinpointing protein locations in the complex cellular tapestries that underpin human health and disease.

Primary Antibodies: How To Check Reliability

One of the most important components for accurate and reliable high-quality immunofluorescence staining is the specificity of the primary antibody to its target antigen, regardless of whether it is fluorophore-conjugated or not. This is absolutely critical to ensuring that any results obtained aren’t due to off-target binding or non-specific autofluorescence and that conclusions are robust and reproducible.

One surefire way to ensure a primary antibody provides a reliable signal is to check the datasheet provided by the manufacturer for images showing expected staining patterns.

At Antibodies Inc., many of our primary antibodies are knockout-validated to show that no signal is detected upon removal of a protein by genetic knockout or knockdown. This clearly demonstrates that the antibody is specific for the target antigen with little off-target binding. Where an antibody is knockout validated, this is indicated on the product page, and data is provided in the specific datasheet.

While internal validation is beneficial, it is no match for independent verification. Therefore, the independent use of a primary antibody in peer-reviewed studies is perhaps the most powerful indication of how robust an antibody might be.

Because of this, alongside its knockout validation status, we also include the number of citations each primary antibody has received on each product page. For instance, our Anti-PSD-95 antibody (75-028) is knockout-validated and independently cited over 800 times for immunofluorescence and numerous other applications in various species. With this information, you can be confident that the antibody you choose is the best for the job at hand.

Another consideration in choosing an antibody is whether it is monoclonal or polyclonal. Monoclonal antibodies bind to only one epitope, providing high specificity and good affinity, whereas polyclonal antibodies recognize multiple epitopes within a given protein which can produce a more robust signal especially for lowly expressed proteins. One major concern around polyclonals is batch inconsistency; we have overcome this issue for our rabbit polyclonal range (PhosphoSolutions) through our unique serum pooling initiative, whereby all bleeds collected from all animals are screened. All bleeds containing high quality antibody are then pooled to ensure all lots are subsequently purified from identical aliquots of homogenous starting material resulting in an abundant supply of antibodies, delivering consistent performance for decades. Furthermore, for the chicken polyclonal antibodies (Aves Labs), one chicken can produce vast quantities of IgY in egg yolk against even low amounts of antigen. Compared to rabbits, chickens can produce up to 20 times more antibodies, making them highly efficient, convenient, and straightforward to produce in a matter of weeks, thus providing a lifetime of material for scientific research.

Finding a reliable primary antibody for immunofluorescence is only the first step to a successful experiment. Upon purchasing a new antibody, checking that it works as expected in your specific cell type or experimental conditions is crucial. Similarly, despite the relative simplicity of immunofluorescence staining, the workflow is prone to experimental errors, which can lead to incorrect interpretations of images and findings that are difficult to reproduce.

Therefore, to ensure antibody specificity under your experimental conditions and avoid frustrating troubleshooting, it is essential to include key controls in each immunofluorescence experiment.

Essential Controls in Immunofluorescence Staining

Including the most appropriate controls in your experiment will help determine the specificity of the primary antibody for a particular antigen or any secondary antibody cross-reactivity. This is central to correctly interpreting staining patterns and any subsequent conclusions made.

For instance, some tissues, such as the brain, lung, and colon, can suffer from extensive autofluorescence due to high levels of elastin, collagen, or lipofuscin. Using only the primary antibody and omitting the secondary antibody can indicate this level of background fluorescence. In experimental samples containing both primary and secondary antibodies, researchers can then use this background level to determine any signals most likely due to antigen binding.

Similarly, omitting the primary antibody or including an antibody of the same isotype (e.g., IgG2b) is also very useful in determining if the secondary antibody has any cross-reactivity with non-specific targets. Any signal observed will likely be due to the non-specific binding of the secondary antibody.

One of the more difficult but most powerful controls to determine the specificity of a primary antibody is to include a positive control. This could be cells where the protein is known to be expressed or artificially overexpressed from a transgene or plasmid. If more signal is observed in specific locations in these overexpressed samples that are absent from negative controls, it’s a good indication that the antibody is specific.

For an even greater level of certainty that the primary antibody recognizes the correct targets, knockdown or knockout samples where the protein of interest is completely removed from a sample also provide a powerful read-out of antibody specificity, especially when combined with samples where the protein is overexpressed. Antibodies Inc. has knockout-validated many primary antibodies to give the confidence that they are specific.

Also, don’t forget to try the antibody at different dilutions upon first use. 1:500 or 1:1000 are common dilutions, but the most appropriate concentration will depend on the cell type, tissue, or staining protocol used.

In addition to checking antibody specificity and including the correct controls, things get a little more complicated when assessing multiple proteins in the same sample, especially with indirect immunofluorescence.

Of special note, visualizing post-translational modification events in immunofluorescence is essential to deduce the biological function of a protein. The PhosphoSolutions range has been extensively validated to ensure the antibodies recognize specifically the target phosphorylation site. For instance, our Anti-ERK/MAPK (Thr202/Tyr204) Antibody has been validated for use in immunofluorescence, and demonstrated to be phospho specific through using relevant controls and simulated samples.

Considerations for Multiplex Immunofluorescence Experiments

Researchers often not only want to pinpoint the exact location of a single protein within a cell but also want to determine how its location relates to another protein of interest by labeling them with different colors. Direct immunofluorescence is the easiest option if the desired fluorophore-conjugated primary antibodies are commercially available.

If they’re unavailable, indirect immunofluorescence must be used, and the different primary antibodies must be derived from another species to allow specific fluorescent labeling with species-specific secondary antibodies.

For instance, if you are interested in “protein X” in human cells, this could be recognized by an anti-protein X primary antibody raised in mouse to the human protein. The secondary antibody could then be an anti-mouse antibody raised in goat, conjugated to a 490 nm fluorophore (green).

To detect “protein Y” in the same sample, you could use an anti-protein Y primary antibody raised in a species other than mouse, like rabbit. The secondary antibody could then be an anti-rabbit secondary antibody raised in goat but conjugated to a 594 nm fluorophore (red).

After imaging on a microscope, the red and green labeled structures would accurately indicate the relative locations of protein X and protein Y in the cell or tissue sample, with yellow labeling representing the presence of the two proteins in the same structure, such as neurons or synapses.

For immunofluorescence multiplexing, our chicken polyclonal range (Aves Labs) is extremely popular as these antibodies show exceptionally high sensitivity and specificity, with no cross-reactivity to mammalian-derived antibodies (please see ‘The Power of Polyclonal Chicken Antibodies in Multiplex Immunofluorescence’). Furthermore, different mouse monoclonal isotypes also make ideal tools for multiplexing (Getting more out of your mouse monoclonal antibodies – multiplex staining with subclass specific secondaries).

The Power of Immunofluorescence in Human Disease Studies

One great example of how a high-quality, well-validated, and cited antibody can be used in multiplex immunofluorescence studies to provide insight into human disease is our monoclonal anti-PSD-95 antibody (K28/43) available in unconjugated or fluorophore-conjugated format.

PSD-95 is a crucial synaptic scaffold protein involved in forming and maintaining synaptic junctions (Keith and El-Husseini, 2008). Thanks to the precise location of PSD-95 in the synapse, studies routinely use our anti-PSD-95 antibody as a clear synaptic marker to determine if other proteins are also present in the synapse.

For instance, researchers recently asked if shorter variants of two key brain receptor proteins called GluN2A and GluN2B found in neuropsychiatric disorders remained at the synapse in hippocampal neurons (Kysilov et al, 2024).

They did this using immunofluorescence staining to assess the level of overlap of the PSD-95 signal in the synapse with the GFP-tagged GluN2A/B proteins. The researchers found significantly less signal from the disease-associated versions colocalizing with PSD-95 at the synapse than the healthy version. This potentially paves the way for the development of new therapeutics. Without the anti-PSD-95 antibody, its specificity, and its localization at the synapse, this and countless other advances could not have been made.

Overall, the hints and tips in this brief guide should arm you with the knowledge and the tools to perform robust, reliable, and reproducible immunofluorescence experiments that illuminate the complex molecular world underpinning human health, development, and disease.

For more information, please get in touch here or visit our product pages.

References

Coons, A.H., Creech, H.J. and Jones, R.N., 1941. Immunological properties of an antibody containing a fluorescent group. Proceedings of the society for experimental biology and medicine, 47(2), pp.200-202.

Keith, D.J. and El-Husseini, A., 2008. Excitation control: balancing PSD-95 function at the synapse. Frontiers in molecular neuroscience, 1, p.200.

Kysilov, B., Kuchtiak, V., Hrcka Krausova, B., Balik, A., Korinek, M., Fili, K., Dobrovolski, M., Abramova, V., Chodounska, H., Kudova, E. and Bozikova, P., 2024. Disease-associated nonsense and frame-shift variants resulting in the truncation of the GluN2A or GluN2B C-terminal domain decrease NMDAR surface expression and reduce potentiating effects of neurosteroids. Cellular and Molecular Life Sciences, 81(1), p.36