Based on the continuous development of fluorophore chemistry and the development of increasingly sensitive instruments, it is now possible to have a fluorescence readout for almost any immunoassay application. While immunofluorescence techniques can provide extremely sensitive and accurate results and are well suited for multiplexing, rational interpretation of the data generated requires thorough optimization by researchers.
A typical immunofluorescence staining protocol involves a series of defined steps, i.e. fixation/permeabilization (if using cell or tissue samples), blocking, primary antibody incubation, washing, incubation with fluorophore-labeled secondary antibody, further washing, and then detection read out. Since most researchers perform repetitive staining protocols, such as measuring effects on expression levels of specific proteins under different conditions and in different tissues, it is recommended to spend time optimizing each step of the protocol before generating data from precious sample material.
For primary antibody incubations for TMCT cell staining, the recommended dilution of the antibody must always be considered to achieve a high signal-to-noise ratio. At very low antibody concentrations, the fluorescent signal may be weak and therefore difficult to distinguish from background, but if the antibody concentration is too high, higher background staining may result. Titrating the primary antibody concentration is key; if concentrations above 10 µg/mL are required, this may indicate poor affinity for the target, or the antibody is not suitable for the application.
Although many modern fluorophores are relatively photostable, failure to protect fluorophore-antibody conjugates from light during storage and staining protocols may degrade them, leading to false-negative results. Fluorescent substances must be carefully stored at recommended temperatures and kept in the dark at all times to protect their spectral integrity. It is important to remember that tandem fluorophores may be particularly sensitive to photodegradation or instability caused by frequent handling.
Proper storage of sample material is also wise. While the immunofluorescence technique provides accurate results, disadvantages to be aware of include autofluorescence, quenching of the fluorescence signal upon excitation, and attenuation of the signal when the sample is stored at ambient temperature. By following recommended sample and reagent handling protocols, researchers can avoid some of these pitfalls of immunofluorescence techniques.
A major advantage of immunofluorescence over immunoenzymatic techniques is that it provides ample opportunities for multiplexing, and flow cytometry is now capable of routinely measuring more than 20 discrete parameters per cell. Without fluorophore-conjugated antibodies, most high-content data cannot be obtained at all, but accurate data analysis depends on careful selection of fluorophores.
When designing multiplexing experiments, due consideration should be given to the unique properties of each fluorophore. Factors such as maximum absorption and emission maximum wavelengths, extinction coefficients, and Stokes shifts should be considered. Often multiple antigen detection is required in a single tissue sample or cell mixture, and selection of fluorophores with non-overlapping spectral distributions is critical. Even the best detection systems cannot overcome the mismatch between the optical properties of the instrument and the fluorophore.
Analysis of any experimental data relies on inclusion of relevant controls, which of course applies to immunofluorescence staining. Omitting the primary antibody will reveal whether any observed signal is due to non-specific secondary antibody binding to moieties in the tissue or cell sample. In addition, cells or tissues known not to express the target should be included as negative controls – eg, those in which the target has been knocked out. In contrast to MAP2 cell staining, to ensure that all components of the assay protocol function as expected even when no signal is observed, cells or tissues known to express the target antigen should be included as positive controls. In techniques such as fluorescent western blotting or ELISA, purified proteins or peptides containing the antigen of interest can provide an appropriate positive control.
Immunofluorescence staining represents a popular and extremely powerful detection method. Through careful selection of fluorophores and development of robust staining protocols, large amounts of data can be generated.
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