Ten Tips to Light Up Your Immunofluorescence Experiments

Immunofluorescence (IF) is a microscopy-based technique that utilizes fluorescently labeled antibodies to visualize a target protein in a sample. Using a combination of various antibodies, a broad array of targets can be detected simultaneously, enabling a high-resolution picture of the presence and localization of different cell populations or proteins in a cell culture or tissue sample.

Recently, advances in fluorophore production and microscope development have increased the capabilities of multicolor fluorescence imaging. The possibilities available with IF may seem overwhelming, but don’t worry! We’ve got your back.

In this blog, we have compiled our top ten tips to assist you with the selection of fluorophores for your imaging experiments.

1. Get to know your microscope — in particular, the emission/excitation/dichroic filters and, in the case of confocal microscopes, the laser lines. Knowing your setup ensures that you can select fluorophores that can be optimally excited and detected. We recommend you consult a spectraviewer or a fluorophore reference chart to assess your fluorophore/microscope compatibility. These references help to determine the maximum excitation and emission wavelengths of the fluorophore-conjugated antibodies in your lab. Some dyes, such as Alexa Fluor 488, have been named/numbered according to their approximate excitation maxima (in nm), so these numbers can provide rough guidance for laser and filter selection.

2. Select fluorophores with high extinction coefficients (ε) — one defining factor of a fluorophore’s brightness is its extinction coefficient (a measurement of the probability of absorbing a photon of light); the higher the value, the brighter the fluorophore. For example, the rather dim DyLight Fluor 350 has an extinction coefficient of only 15,000 while the bright DyLight Fluor 650 dye has an extinction coefficient of 250,000.

3. Choose fluorophores with high quantum yields (Φ) — the quantum yield is a read-out of the efficiency of the fluorescence process, calculated by dividing the number of emitted photons by the number of absorbed photons. A 100% efficient fluorescence process would have a quantum yield of 1, the maximum possible. The commonly used, bright Alexa Fluor 488 fluorophore, for example, has a high quantum yield of 0.92.

4. Avoid fluorophores with a high susceptibility to photobleaching — to reduce fading of the fluorescence signal, we recommend you use photostable fluorophores, such as StarBright Dyes, Alexa Fluor, or DyLight Fluor dyes. FITC and R-phycoerythrin (RPE) are known to photobleach quickly. Alternative options to minimize photobleaching are to reduce the intensity/exposure time to the excitation light and to use mounting media containing antifade reagents.

5. Use new generation dyes that stay fluorescent over a broad pH range — many conventional fluorophores, such as FITC, are not recommended for staining protocols using acidic buffers, as the fluorescence intensity signal is highly sensitive to an acidic environment (Chen et al. 2009).

6. Use smaller, more stable, single fluorophores — tandem dyes, such as PE-Cy5 and APC-Cy7, consist of two linked fluorophores. The energy emitted upon excitation of the donor fluorophore excites the acceptor fluorophore, allowing a single laser to excite multiple fluorophores. However, despite their versatility, they present several challenges:

• Poor cell penetration due to large molecular size
• Chemical instability — susceptible to degradation from light, fixation reagents, or prolonged storage
• Lot-to-lot variability affecting emission spectra
• Broad emission spectra causing channel overlap and complicating multicolor imaging

Additionally, large fluorophores, like PE, can sterically hinder antibody binding, especially in areas with densely packed epitopes. Using smaller fluorophores or quantum dots improves binding efficiency and staining uniformity.

7. Ensure your fluorophore staining is spectrally differentiable from your counterstain — the counterstain provides background contrast and puts the observed staining into perspective (e.g., by visualizing nuclei). For example, DRAQ5 should be used as a nuclear counterstain rather than DAPI when using antibodies conjugated to blue-emitting fluorophores, such as Alexa Fluor 405 or DyLight Fluor 405.

Table 1. Some commonly used counterstains and their respective emission colors. Maximum excitation and emission data from FluoroFinder.

Counterstain	Color	Target	Max Excitation (nm)	Max Emission (nm)
DAPI	Blue	Nucleus	350	465
DRAQ5	Red	Nucleus	647	681
Hoechst 33258/33342	Blue	Nucleus	352/361	454/497
Phalloidin	Variable, depends on the conjugated dye	Filamentous actin	Variable, depends on the conjugated dye	Variable, depends on the conjugated dye
Propidium iodide	Red	Nucleus	535	617
Wheat germ agglutinin (WGA)	Variable, depends on the conjugated dye	Plasma membrane	Variable, depends on the conjugated dye	Variable, depends on the conjugated dye

8. Use fluorophores with narrow emission spectra — this reduces the likelihood of detecting signal from one fluorophore in another's filter set, thus minimizing spectral overlap. Spectral overlap makes it difficult to observe discrete fluorescence signals and complicates the evaluation of colocalization experiments. It is advisable to use a spectraviewer to check for overlap. Ideally, there should be no spectral overlap between the fluorophores. Quantum dot conjugates, which have broad absorption and very narrow emission spectra, are therefore well suited for multicolor experiments. Multiple antibodies conjugated to quantum dots can simultaneously be detected with negligible spectral overlap (Olympus 2015).

9. Carefully decide what antigen to detect with which fluorophore — the brightest fluorophore should be reserved for the detection of the antigen with the least abundant expression level. The dimmest fluorophore should be used for detecting the most abundant antigen.

10. Include appropriate controls to verify your fluorophore staining — we recommend you consider these three controls:

• Autofluorescence/cell background staining control. Because cells are naturally fluorescent, it is important to observe your IF samples microscopically after fixation and blocking, but before staining. Additionally, an unstained control should be included, which, as the name suggests, is a sample that has not been stained with any antibody
• Positive and negative controls. Include cell lines in which your protein of interest is either overexpressed or absent (e.g., a knockout cell line). If you do not see staining in the positive control, something has gone wrong with the staining protocol. Alternatively, if you see staining in the negative control, you know that the staining/observed fluorescence is nonspecific
• Isotype control. Isotype controls are used to rule out nonspecific binding of the primary antibody, for example, binding of the antibody by Fc receptors found on immune cells. The isotype control will be generated in the same host species, with the same isotype and conjugate, but with a different target specificity
• If you are using secondary antibodies rather than directly fluorophore-conjugated primary antibodies, a secondary-only control should be performed (following the same staining protocol without the addition of a primary antibody). This is used to verify that the secondary antibody does not nonspecifically bind to certain cellular compartments. For multicolor IF experiments, the use of cross-adsorbed/pre-adsorbed secondary antibodies is recommended, as those minimize the risk of the secondary antibody reacting with endogenous immunoglobulins or an undesired primary antibody

By keeping these tips in mind for your IF experiments, we hope you feel empowered to light up the dark unknown and illuminate the answers to your research questions.