Thanks to the continuous evolution of fluorophore chemistries and the development of increasingly sensitive instruments, almost any immunodetection application can now have a fluorescent readout. Yet it should be remembered that while immunofluorescence techniques can afford extremely sensitive and accurate results and are highly amenable to multiplexing, sensible interpretation of the data that is generated requires that researchers perform thorough optimization.
Chandra Mohan, Ph.D., senior manager, scientific content and training, and Robin Clark, Ph.D., content development & antibody validation scientist, at MilliporeSigma share their expertise for successful immunofluorescent staining in the tips below.
A typical immunofluorescent staining protocol involves a defined sequence of steps, namely fixation/permeabilization (if working with cells or tissue samples), blocking, primary antibody incubation, washing, incubation with a fluorophore-labelled secondary antibody, further washing, and then the assay readout. Clark explains that “since most researchers will perform repetitive staining protocols, such as those where the effect on expression levels of a particular protein is measured under different conditions, and in different tissues, it’s advisable to spend time optimizing each step of the protocol before generating data from precious sample material.”
Mohan points out that “In terms of the primary antibody incubation, for example, one must always consider an antibody’s recommended dilution to achieve a high signal-to-background ratio. At very low antibody concentration the fluorescence signal may be weak and therefore difficult to distinguish from the background, yet if an antibody is used at excessive concentration it may create higher background staining. Clark adds that “titrating primary antibody concentration is key; if concentrations over 10 µg/mL are required, this may indicate poor affinity for the target, or inappropriateness of the antibody for the application.”
Image: Immunofluorescent staining of TMTC3 using a Anti-TMTC3 antibody from MilliporeSigma.
Clark also says that the importance of thorough washes between steps should never be underestimated. “Washing in physiological solutions with at least one buffer change per wash step will enhance signal-to-background ratio by removing unbound fluorescent reagents, or those that are merely sticky and not specifically bound to the target.”
Despite the relative photostability of many modern fluorophores, failure to protect fluorophore-antibody conjugates from light during storage and staining protocols can degrade them, leading to false negative results. Clark emphasizes that “fluorescent species must be stored carefully at the recommended temperature and kept in the dark at all times to protect their spectral integrity. It is important to bear in mind that tandem fluorophores can be particularly sensitive to photodegradation or instability induced by frequent handling.”
Mohan adds that it is also wise to store sample material appropriately. “While immunofluorescence techniques provide accurate results, drawbacks to be aware of include autofluorescence, quenching of fluorescent signal at excitation, and fading of signal upon storage of specimens at ambient temperature. By following recommended protocols for specimen and reagent handling, researchers may avoid some of these immunofluorescence technique pitfalls.”
Image: Immunofluorescent staining of SPRYD4 using an anti-SPRYD4 antibody from MilliporeSigma.
A major advantage of immunofluorescence over immunoenzymatic techniques is that it affords ample opportunity for multiplexing, with flow cytometrists now able to routinely measure more than 20 discrete parameters for every cell. “Most of this high-content data simply could not be achieved without fluorophore-conjugated antibodies,” says Clark, “yet accurate data analysis depends on careful fluorophore selection.”
When designing a multiplexing experiment, the unique properties of each fluorophore should be afforded due consideration. Factors such as the maximal absorption and maximal emission wavelengths, extinction co-efficient, and Stokes shift should all be taken into account. “Often there is a need for multiple-antigen detection in a single tissue sample or mixture of cells,” says Mohan, “and it’s essential to select fluorophores with non-overlapping spectral profiles. Even the best detection systems cannot overcome mismatches between instruments and fluorophore optical characteristics.”
Analysis of any experimental data relies on the inclusion of relevant controls, and this of course holds true for immunofluorescent staining. “Omission of the primary antibody will reveal whether any observed signal is due to nonspecific secondary antibody binding to moieties in the tissue or cell sample,” explains Clark. “In addition, we recommend that as a negative control cells or tissue known not to express the target should be included—for example, those where the target has been knocked out. Conversely, to ensure that all components of the detection protocol are functioning as expected even when no signal is observed, cells or tissue known to express the target antigen should be included as positive controls. In techniques such as fluorescent Western blotting or ELISA, the purified protein or peptide containing the target antigen provides an appropriate positive control.”
Image: Immunofluorescent staining of MAP2 using a monoclonal anti-MAP2 antibody from MilliporeSigma.
Immunofluorescent staining represents a popular and extremely powerful detection method. Through careful fluorophore selection and development of a robust staining protocol it is possible to generate a wealth of data. MilliporeSigma offers high-quality reagents and technical expertise to help you achieve your immunofluorescent goal. Learn how to further optimize your immunofluorescence experiments at SigmaAldrich.com/antibodies.