The Immunofluorescence (IF) is a useful technique for the detection and localization of cellular antigens using antibodies labeled with fluorochromes. Although the procedure is relatively simple, including the steps of fixing and permeabilizing the samples, blocking and incubation with the labeled antibodies, in many cases the success of the assay may depend on the correct adjustment of certain variables during the process.
In this post we collect some tips that will allow you to optimize your Immunofluorescence (IF) experiments.
1.- Sample Preparation: Fixation And Permeabilization
These steps are essential for the antibodies to access the target antigen, and optimization of the variables is critical so as not to affect cellular integrity and that of the antigen itself.
The objective of this process is to preserve to the maximum the cellular morphology with respect to its native state. For this, there are two large groups of fixing reagents, each with its advantages and disadvantages: aldehydes and organic solvents.
- Aldehydes : they correctly preserve cell morphology, and are especially recommended for the visualization of membrane proteins. In counterpart, the antigenicity of the target protein may be reduced.
- Organic solvents : they correctly preserve the cellular architecture, and add the advantage of not requiring a subsequent permeabilization step. However, they have some drawbacks such as a large amount of lipids and small soluble molecules are removed during the fixing process.
This step will only be necessary in the case of having used aldehydes as a fixing agent.
2.- Buffers And Blocking Agents
Although the buffer of choice is usually PBS, since being isotonic it does not alter the cellular structure and maintains the pH at levels close to physiological, in some occasions when a weak signal is obtained, it will be necessary to try other alternatives with different ionic compositions of calcium, magnesium and potassium.
- BLOCKING AGENTS
The blocking step is necessary to avoid non-specific binding of the antibodies. Although the most common is BSA, in certain cases it is convenient to try other alternatives such as fetal bovine serum, casein or gelatin to try to optimize the signal.
Antibodies are one of the most critical reagents in immunofluorescence experiments.
The antibodies used in immunofluorescence assays must be highly specific against the antigen of interest, although this does not necessarily imply that they must be monoclonal.
For example, in those cases that require high precision such as the labeling of the c-terminal end of a certain protein, the use of monoclonal antibodies is recommended . However, in cases where a higher affinity is required, such as when the protein is present in very low concentrations, polyclonal antibodies will be the most indicated.
Remember this post on the differences between monoclonal and polyclonal antibodies for more information.
To optimize the staining, it is always recommended to titrate the antibodies by serial dilutions, to opt for the concentration that allows to improve the signal intensity, keeping the background noise low.
- Secondary antibodies
In case of performing an indirect immunofluorescence (IIF) experiment, we must also pay attention to the choice of secondary antibodies (remember this guide to select secondary antibodies). These antibodies should react not only against the species in which the primary antibody originated, but also against its isotype.
In order to minimize cross-reactivity, especially in multi-color experiments where several primary antibodies and their corresponding secondary antibodies are used simultaneously, it is recommended to carry out an additional pre-adsorption step of these secondary antibodies, passing them through a column where the serum proteins of those species with which there is a risk of cross-reaction have been previously immobilized.
4.- Selection Of Fluorochromes
To select the fluorochrome that best suits our experiment, we must assess several factors:
- Characteristics and functionalities of the microscope : we must ensure that the selected fluorochromes can be optimally excited and detected.
- Fluorochrome Characteristics
– Extinction coefficient : the higher the extinction coefficient, the brighter the signal it emits.
– Quantum performance : it is an indicator of the performance of the fluorescence process, therefore, the ideal would be to opt for fluorochromes with high quantum performance.
– Susceptibility to photobleaching : the use of photostable fluorophores is recommended, so that the intensity of the signal is not reduced by a process of photochemical destruction.
– Counter staining : it is necessary to ensure that the spectrum of fluorochrome is different from that of counter staining, which will facilitate background contrast.
To contextualize the specific signal of our sample, it is necessary to use counterstains against cellular structures such as the nucleus, the cytoskeleton or the plasma membrane. Unlike antibodies, counter stains do not react with each other and can be incubated at the same time in a single step.
As we have seen, there are several methods to fix, permeabilize and stain cells, each presenting its advantages and disadvantages. The Immunofluorescence (IF) protocol should be optimized in each specific case, according to these criteria, and based on the specific target that we intend to analyze and its location.