Confocal image of cardiomyocytes (rat) showing the actin cytoskeleton in red, microtubules in green, and the myofibrils in blue. The bottom-middle cell is a fibroblast that lacks expression of sarcomeric alpha-actinin and hence has no blue striations.
Elisabeth Ehler’s office walls at the Randall Centre for Cell and Molecular Biophysics at Kings College London display the results of her team’s work over the years: journal covers featuring sharp images of cardiomyocytes. But even her experienced group of cell biologists needs different multiple labelling protocols to meet the challenges of each project.
When using new antibodies, Ehler’s team has to adapt protocols to ensure the best results. On one occasion, this involved incubating the cells overnight in the fridge as opposed to room temperature.
Optimizing the incubation conditions is just one of Ehler’s strategies. She has several tips for researchers who are trying to improve their immunofluorescence skills and create high-quality, detailed images of subcellular processes – even when labelling multiple targets at once.
Test reagents and equipment
Immunofluorescence microscopy uses a primary antibody to bind the target of interest, and a secondary antibody to bind to that complex. The secondary antibody comes conjugated with a fluorophore (or fluorochrome) that is used to visualize the location of this complex in the cell. In a multiple labelling experiment, there are several primary antibodies, each recognizing a different target, and each with its own secondary antibody. The number of targets per experiment is usually limited by the number of channels available in the microscope.
Ehler tests her primary antibodies before starting. “I always do a Western blot with tissues where I know that the target protein is either highly expressed or not at all.” This confirms the specificity of the reagent. She also tests a range of different dilutions; too concentrated and there is a risk that the antibodies form unspecific reactions, but too dilute and the signal is barely visible.
Choice of secondary antibody conjugate is crucial. Ehler recommends first checking which wavelengths the microscope can detect. “You can have the best antibodies and the best microscope,” she says. “But if the filters on your microscope are not optimal for your antibody’s fluorochrome, or if the laser doesn't excite that wavelength, you’re not getting the best image.”
It is also important that the fluorophores’ emission spectra don’t overlap. If the wavelengths are too close together, the signal from one may bleed through to another channel. To avoid this, Ehler prefers to visualize them sequentially.
Confocal image of mouse diaphragm showing alternate M-bands and Z-discs in the muscle fibres. Alpha-actinin is stained blue; M-protein is green and myomesin is red, which are both localized in the M band of the sarcomere, the basic unit of a myofibril, hence the yellow signal when co-expressed in muscle fibres.
Reducing cross-reactivity
When using several secondary antibodies, they should all originate from the same species to reduce the likelihood that they will react with each other. Ehler also uses serum in her blocking buffer from the same species as the labelled antibody.
Secondary antibodies optimized for multiple labelling are cross-adsorbed to ensure that there is no interaction with an unintended primary or with the sample cells. Ehler recounts an instance where a lab member tried a new antibody that wasn’t optimized for the job they were doing. The anti-rabbit antibody had some residual cross-reactivity against mouse, which appeared as unexpected colocalizations in the final images. The team now only buys the correctly cross-adsorbed antibody for each experiment.
Howard Damude is familiar with this type of problem. He is a senior scientist at secondary antibody manufacturer Jackson ImmunoResearch in West Grove, Pennsylvania. “The single most critical criterion is to ensure that each of the secondary antibodies has minimal cross-reactivity to antibodies from other species being used, and to the species from which the sample comes,” he says.
Damude describes the strict quality control of Jackson ImmunoResearch’s antibodies, particularly those for multiple labelling, where cross-reactivity is removed with affinity chromatography. “We pride ourselves on delivering very high-quality reagents,” he says. Damude also encourages researchers to ask for help. Jackson ImmunoResearch can answer questions and provide advice before researchers start an experiment, including the most challenging, multi-labelling experiments.
Ultimately, says Ehler, when it comes to immunofluorescence micrographs: “Your success will be defined by the quality of the antibodies.”