How many samples? How much money and time have you wasted trying to unmask a protein on your IHC samples? Tissue fixation in immunohistochemistry often negatively impacts your immunostaining by masking the epitope of interest. Read on to learn about the most common antigen retrieval methods used to restore epitope-antibody binding.

Fixation of a tissue sample by immunohistochemistry is required if you want to preserve tissue morphology and retain the antigenicity of the target protein during your experiment. In performing their protective role, fixatives either cross-link proteins as with aldehydes such as formaldehyde or denature proteins by coagulation, like with alcohols such as methanol. Heating your tissue in a high or low-pH buffer or enzymatically digesting it with proteases can do the trick by unmasking the epitopes recognized by the antibody.

The masking effects

The masking effects of antigens by chemical fixation represent a problem for immunohistochemical purposes. Although formalin is a cross-linking type of fixative, forming methylene bridges between proteins within the tissue, is still the fixative of choice and has been used since 1893 as the standard fixative for tissue processing in histopathology. 

The major artifact induced by fixation is the masking of tissue antigens due to cross-linking among the amino-acid residues of proteins. This cross-linking reaction adversely alters the structure of tissue proteins, resulting in the loss of antigenicity, that is the ability of the primary antibody to recognize the peptide epitope.

The observation that the cross-linkages could be reversed by high-temperature heating or strong alkaline treatment, formed the basis for the development of antigen retrieval techniques in 1991. Today different approaches to antigen retrieval exist.

How to retrieve the masked antigen?

The introduction of antigen retrieval methods in the early 1990s boosted a revolution in the practice of pathology and immunohistochemical research. For the first time, it became possible to reveal a large variety of proteins in formalin-fixed, paraffin-embedded (FFPE)-tissues. 

Antigen retrieval is a rather simple laboratory technique that has become an essential part of any IHC-staining procedure for FFPE tissues. An antigen retrieval treatment is applied to unmask the epitopes, either by heat (heat-induced epitope retrieval, HIER) or enzymatic degradation (proteolytic-induced epitope retrieval, PIER) or through a combination of both approaches (infrequently used).

The purpose is to remove the protein cross-links formed by formalin fixation. This process allows the antigens that are difficult or impossible to stain to become readily stainable by standardized immunohistochemical methods.

As a general rule, antigen retrieval procedures involve heating slide-mounted specimen material in a buffer solution. Heat causes cross-linked protein epitopes to unfold in a manner similar to DNA denaturation, while buffer solutions aid in maintaining the conformation of the unfolded protein.

1. PIER: the chemical approach for epitope retrieval

It makes use of enzymes to retrieve antigens and restore antigenicity. Enzymes degrade protein crosslinks. It is usually performed at a temperature of around 37°C. Common buffers used in PIER are neutral buffer solutions of enzymes such as Trypsin, Proteinase K, Pepsin, Protease, and Pronase.

Disadvantages of the PIER antigen retrieval method include destroyed tissue morphology, and degradation of the epitope causing falsely negative results. Under-digestion (resulting in too weak immunostaining, since the antigens are not fully exposed). Over-digestion (can produce false-positive staining, high background levels, and tissue damage).

2. HIER: the physical approach for epitope retrieval

Implies the use of heat to retrieve antigen and restore antigenicity usually performed at over 50°C. Heat causes the crosslinked protein to unfold. The buffers' composition depends on the pH required which is target-dependent. Popular buffers include low pH citrate buffer solution (pH 6.0) and high pH Tris-EDTA (pH 8.0-9.9)

Listed below are different devices that can be used for HIER. In all cases, the temperature achieved by these methods and the pH of the antigen retrieval solutions appears to be the critical variable.

Antigen retreival: PIER or HIER?

Both enzymatic (PIER) and non‐enzymatic (HIER) antigen unmasking are not dependent on the epitope sequence, but some antigens benefit selectively from one treatment but not from the other.  

In general, HIER has a higher success rate than PIER. The latter method acts by unmasking the peptide epitope(s) enzymatically and may also alter the morphology of the specimen or the antigen itself. Consequently, PIER is less frequently used than HIER, which rather acts by restoring the secondary and tertiary structure of an epitope. 

However, enzymatic digestion by PIER is sometimes suggested by a manufacturer. To determine the ideal antigen retrieval solution, I recommend using a “test battery,” because different antigens may require different conditions for retrieval.

Figure 2. Different antigen retrieval conditions lead to different staining results. This example shows immunohistochemical staining of Zinc finger C4H2 protein in human smooth muscle tissue with a polyclonal antibody targeting ZC4H2. Expected membranous staining.

Is antigen retrieval always necessary?

No, it is not. The need for antigen retrieval depends a lot on your specific antigen and the primary antibody you will use to immunolabel it. You may need antigen retrieval to obtain any labeling at all or to improve your staining and make it more consistent.

Some antigens, being very robust, abundant, or resilient to strong fixation still yield good immunolabeling. Antigen retrieval is usually not required for immunostaining of fresh frozen sections (they are usually sectioned and fixed with ice-cold acetone for 5-10 minutes).

Antigen retrieval methods depend on your specific antigen and the primary antibody you will use to immunolabel it. So here is a list of simple yet useful tips:

• Always review the literature to look for the antigen retrieval methods suggested for the protein you are interested in.
• If no specific method is indicated for your specific protein, start testing HIER at low and high pH and PIER (different enzymes).
• To eliminate staining artifacts created by the HIER process, compare it against a control sample for which no HIER treatment was performed.
• Make a proper investment in a good heating device for HIER.

Readings

Shan-Rong Shi, Yan Shi, and Clive R. Taylor (2011) Antigen Retrieval Immunohistochemistry Review and Future Prospects in Research and Diagnosis over Two Decades. J. Histochem Cytochem. 59(1):13–32.

Shan-Rong Shi, Richard J. Cote, Clive R. Taylor (2001) Antigen Retrieval Techniques: Current Perspectives J. Histochem Cytochem. 49(8):931-937

Shan-Rong Shi, Richard J. Cote, Clive R. Taylor J (1997) Antigen Retrieval Immunohistochemistry: Past, Present, and Future J. Histochem Cytochem. 45 (3): 327-343

Shi S-R, Key ME, Kalra KL. (1991) Antigen retrieval in formalin-fixed, paraffin-embedded tissues: An enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J. Histochem Cytochem. 39(6):741-74