Antigen Retrieval Methods

IHC Antigen Retrieval

At times the conditions of tissue fixation will mask the antigen and prevent it from being recognized by the antibody. Most often, this is the result of cross-linking proteins that have transiently changed conformation. This causes the epitope recognized by the antibody to be hidden from view. In this scenario, you may experience a dramatic loss in the ability to detect your protein of interest. There is no need to worry, though, because you can use an antigen retrieval method to troubleshoot the problem.

What Are Different Methods for Antigen Retrieval?

Basically, all methods of unmasking the epitope rely on breaking protein crosslinks. One general category of antigen retrieval is heating. You can use a microwave oven or a steamer to promote breakage of crosslinks. You can also make use of proteolytic methods for a slightly more selective unmasking procedure. Common enzymes used in this application include proteinase K, trypsin, and pepsin.

There are pros and cons associated with both methods. Heat and protein-induced unmasking are both relatively simple. Protein-mediated retrieval is somewhat more gentle that heating. Unfortunately, it can also result in damage to the tissue or to other antigens you hope to detect. Heating can be used to retrieve antigens sensitive to enzyme digestion. It can also lead to non-specific damage in certain tissues, and you run the risk of overheating your sample if you do not control conditions carefully.

How Do I Choose an Antigen Retrieval Method?

The type of unmasking method you use will be determined empirically. It is important to test any antibody that you purchase for different parameters like dilution and binding times. You must also decide what antigen retrieval method is most suitable for that antibody if you need one at all.

Immediately after you purchase an antibody, you must set up an experiment to test optimal conditions. Try a few different buffers with heat-induced unmasking. Also try a few different enzymatic methods. You can then determine the extent to which the retrieval improves the results. A  wide variety of antigen retrieval buffers can be purchased commercially.

If neither method improves the detection by the antibody, then it is likely that it is simply not amenable to paraffin-embedded tissue. Some antibodies are designed only for western blot and do not pick up folded proteins well.


Overall, antigen retrieval is one method you want to keep in mind when troubleshooting IHC. Because there are two different general ways of attacking the problem, you will want to run a well-controlled experiment to determine proper conditions for unmasking.

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