AAT Bioquest

What controls do I need for immunofluorescence experiments?

Posted September 10, 2019


Images obtained from immunofluorescence procedures suffer from two major difficulties: autofluorescence (natural fluorescence of cells in response to the excitation light) and nonspecific antibody binding (when the marker antibody binds to non-target proteins). In order to minimize each of these issues, two controls are required.

The unstained control is intended to help calibrate autofluorescence levels. This control is simply a portion of the sample with no marker antibodies of any kind, allowing for measurement of the natural fluorescence of the cells. Comparison of this control with experimental results will allow for more accurate understanding of any positive readings. If the natural fluorescence of the experimental material is too great, special care must be taken, often by using time-resolved fluorescence (TRF) fluorophores. Another method to minimize autofluorescence is to employ deep IR probes, which use an excitation source at too long a wavelength for most mammalian cell cultures to respond. 

To measure the degree of nonspecific binding, an antibody-isolate control is necessary. This will allow the researcher to determine what degree of antibody binding is occurring with other proteins present in the sample. If the level of nonspecific binding is too great, this suggests that additional optimization or protocol troubleshooting is required. Protocols that include well-prepared washing and blocking steps, as well as correct antibody concentrations, will assist in minimizing undesirable antibody binding. 

For information on antibody selection, conjugation, and optimization, see 'Additional Resources' below. 

Additional resources

Antibody Selection and Detection

Antibodies by Category

iFluor Dyes

Degree of Labeling Calculator