Introduction

With the exception of fresh frozen tissue sections, tissue that has been subjected to fixation, most commonly aldehyde-containing compound such as paraformaldehyde or formalin solutions, shows a partial or complete loss of immunoreactivity due to a phenomenon called epitope masking. Epitopes are distinct molecular surface features of an antigen that are capable of being bound by an antibody.

Tissue fixation works to stabilize tissue and inhibit enzymatic reactions that degrade tissue through forming cross-links and (in some cases) precipitating proteins; for histology, this process produces high-quality morphology at the expense of reducing antibody detection. Since most antibodies are specific for a particular sequence of amino acids in a particular three-dimensional motif, the fixation process that tissue is subjected to, especially when treatments form multiple cross-links, can significantly alter the composition and structure of available epitopes. Consequently, the availability of epitopes to be bound by antibodies are almost always reduced by cross-linking reactions through alterations in secondary and tertiary structures, reducing the overall availability of epitopes that can bound by antibody. In practical terms, tissue sections that never been fixed will almost always demonstrate high levels of reactivity to antibodies, whereas paraformaldehyde or formalin fixed tissue will tend to display low levels of reactivity without antigen retrieval.

Antigen retrieval is a well-established approach that is designed to increase the availability of epitopes that can be bound by specific primary antibody. Although these techniques work very well, and are indeed considered to be a standard approach to achieving high quality staining, the precise mechanisms through which different antigen retrieval techniques work are poorly understood. The investigator should understand that finding an appropriate antigen retrieval method that works with particular antibody should be tested empirically in a systematic process.

Recommendations

As a first approach to determine if an untested antibody will stain a particular marker and tissue, we recommend using fresh frozen tissue to evaluate the quality of staining provided by antibody. Although the morphology that is generated from fresh frozen tissues is inferior to that of fixed tissue, antibodies will typically show the highest degree of sensitivity and staining. Once the investigator established that the new antibody of interest works in tissue, and they wish to test this in fixed tissue, they can systematically test the antigen retrieval methods outlined below to identify the approach that works best.

Antigen retrieval protocols

Heat induced epitope retrieval (HIER)

• An introduction to  HIER techniques and devices
• Citrate Buffer
• Citrate-EDTA Buffer
• EDTA
• Tris-EDTA
• Tris Buffered Saline (TBS)
• Tris Buffer

Room temperature epitope retrieval (RTER)

• Hydrochloric Acid
• Formic Acid

Proteolytic induced epitope retrieval (PIER)

• Proteinase K
• Trypsin
• Pepsin
• Pronase
• Protenase

Frozen section epitope retrieval

• SDS
• Block Heating

Free-floating section epitope retrieval

• Sodium Citrate

Crosslink (Aldehyde) Chemical Modification epitope retrieval

• Citraconic Anhydride