The immunofluorescence (IF) technique, based on the detection of a specific antigen of interest by using fluorescently labeled antibodies, is a technique widely used in research laboratories due to its simplicity and reliability.
The results can be visualized by fluorescence microscopy using short wavelengths and, in addition to detecting the presence or absence of a certain protein in the sample, it is possible to determine its distribution in the sample or confirm the presence of post-translational modifications, among others.
In this post we bring you some keys related to antibodies for immunofluorescence that can help you optimize the results of your tests.
3 KEYS WHEN USING ANTIBODIES FOR IMMUNOFLUORESCENCE
1.- THE IMPORTANCE OF THE SPECIFICITY OF ANTIBODIES FOR IMMUNOFLUORESCENCE
As in any other immunoassay, the specificity of the primary antibody against our target antigen is a determining factor in the reliability and success of the results. The more specific the antibody, the better the signal obtained and the less background noise generated.
Let us also remember that an antibody that has a high specificity against an antigen in a certain technique does not have to do so in another, even if it is the same antigen. Hence the importance of validating each antibody for each technique in which it will be used. In the case at hand, it is essential to previously validate the immunofluorescence antibodies to be used in the assay.
How can we validate the antibodies for immunofluorescence? For there are various methods such as positive and negative expression experiments using, for example, knock-out cell lines, by experimental manipulation of the location of the target protein, protocol optimizations, etc. Or, resorting to commercial antibodies already validated for use in this technique.
2.- CONTROLS FOR IMMUNOFLUORESCENCE
The inclusion of controls, as in any other experiment, will increase confidence in the results obtained in terms of specificity and sensitivity.
To avoid errors derived from autofluorescence phenomena or from nonspecific binding of antibodies, the use of negative controls in immunofluorescence assays is very important.
Additionally, additional controls such as omission of the primary antibody, the use of isotype controls and of negative and positive cell lines for the antigen of interest may be included.
3.- DILUTION OF THE ANTIBODIES FOR IMMUNOFLUORESCENCE
To optimize the results of the tests, another key point is the titration of the antibodies to determine the ideal dilution to use in each case. This will also vary depending on whether we are dealing with a purified antibody or an antiserum.
In this sense, it is important to achieve a good signal / background noise ratio, that is, an optimal relationship between the intensity of the fluorescent signal from the antigen of interest and the background signal due to nonspecific junctions. If we apply the primary antibody at a very low concentration, it will be very difficult to distinguish the positive signal. Conversely, an overly concentrated antibody will excessively increase background noise.
The typical concentration / dilution ranges for immunofluorescence experiments are usually between 1-10ug / mL in the case of using purified antibodies, and between 1: 100 – 1: 1000 for the antisera.
In this post you can remember some other recommended dilutions for other techniques and immunoassays.