Immunofluorescence Troubleshooting

As most researchers know, getting a technique to work in the way it’s supposed to can be a matter of trial and error. Immunofluorescence (IF), a technique that uses antibodies labelled with a fluorescent dye to detect a target antigen, has increased in popularity over the last few decades due to its relative simplicity and reliability. However, problems can sometimes crop up with even the most dependable of methods.

Many common difficulties with IF relate to staining: too much, too little, or in the wrong place. Getting the staining right is crucial, as it allows the researcher to assess both the presence and the distribution of the target antigen in their sample.

There are numerous ways in which incorrect technique or poor laboratory practice can affect staining in IF. Here, though, we will focus on a few of the antibody-related problems that can arise in IF, and suggest some possible solutions.

1. Non-specific staining

Non-specific staining occurs when the primary antibody in IF binds to proteins other than the target antigen, producing results that are meaningless or unusable. In the past, it was suggested that this could have been due to either non-specific antibody binding to endogenous Fc receptors (proteins found on the surface of certain immune cells) or a combination of ionic and hydrophobic interactions; however, both of these ideas have been found to be unsupported. Instead, here are a few of the possible causes of non-specific staining:

Antibody concentration

The concentration of the primary or secondary antibody may be too high, increasing the likelihood of non-specific or excessive background staining. Try reducing the concentration and/or the incubation period (take care not to reduce the concentration too much, however, as the fluorescence signal could then become indistinguishable from background.)

Choice of antibody

If possible, choose a primary antibody that was raised against the same species as the target sample (secondary antibodies should be raised against the host species of the primary antibody.) This will minimise the amount of off-target or non-specific binding. It is also helpful to select an antibody that has previously been shown to perform well in IF. At St John’s Laboratory, we have a catalogue of over 30,000 antibodies to choose from, many of which have been validated in IF applications.

2. High background staining

While a high level of background can be due to non-specific antibody binding, as above, there are also other possible causes, such as autofluorescence – this can be determined by checking an unstained sample for fluorescence. Using an excessively high antibody concentration can also cause problems. Other antibody-related issues resulting in a high level of background include:

Non-specific binding of secondary antibodies

Try running a secondary control without the primary antibody. If there is staining, this suggests non-specific binding is occurring. Consider using a different secondary antibody.

Insufficient/absent blocking

A blocking serum contains antibodies that bind to non-specific sites, reducing the chance of off-target binding of the primary antibody. If high levels of background are appearing, consider either changing the blocking serum or increasing the blocking serum incubation period. Usually, a blocking serum matching the species of the secondary antibody is recommended.

3. Weak or insufficient staining

If a sample is showing an unexpectedly low level of fluorescence, the first step should be to check that the microscope is functioning normally. There could also have been a problem with permeabilisation – the process of treating the sample with chemicals that cause the cell membrane to become permeable to antibodies. If both of these factors are sound, however, there are a few other steps to check:

Antibody concentration/incubation

There might be too little primary antibody available to bind with the target antigen. Try increasing the concentration of the primary antibody and/or the incubation time.

Antibody incompatibility

In secondary IF, the secondary antibody carries the fluorophore. If the primary and secondary antibodies are incompatible, little or no fluorescence will be observed. Choose a secondary antibody raised against the host species of the primary antibody, e.g. if the primary antibody was raised in mouse, select an Anti-Mouse secondary antibody.

Monoclonal versus polyclonal antibodies

Monoclonal antibodies will bind to a single epitope on the antigen, whereas polyclonal antibodies will bind to multiple epitopes. This means that using polyclonal antibodies can boost the signal, as several antibodies should bind to each antigen. However, polyclonal antibodies can also produce a higher level of background.

At St John’s Laboratory, we have a range of over 30,000 monoclonal and polyclonal primary antibodies, as well as more than 50 secondary antibodies. Currently 13,000 of these are available through our Antibody Validation Project, which allows researchers to try sample-size antibodies for free, in return for letting us know how the antibody performs.