IHC Protocol Deep-dive:

Antigen/Epitope Retrieval

Antigen retrieval (AR) has been a revolutionary technique in IHC and has been instrumental in unmasking low-level or formalin cross-linked antibodies. Get more information on epitope retrieval optimization tips in the link below.

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What is Antigen Retrieval?

Antigen retrieval (AR) is the process of breaking protein cross-links that mask antigens in formalin-fixed tissue sections. Formalin or other aldehyde fixation forms protein cross-links that mask the antigenic sites in tissue specimens, giving weak or false negative staining.

What is the purpose of antigen retrieval?

Antigen retrieval breaks the protein cross-links, enhancing staining intensity by unmasking the antigens and epitopes in formalin-fixed and paraffin embedded tissue sections.

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Selecting the right antigen retrieval method

The process of fixing a tissue sample with paraformaldehyde causes the formation of peptide crosslinks that serve to preserve tissue architecture and protein localization. These peptide crosslinks can also mask epitopes, making them inaccessible to antibody binding. In order to get good-quality IHC stains, these cross-links must be broken in the process known as epitope retrieval.

Methods of Epitope Retrieval

There are two common methods of epitope retrieval: heat-induced epitope retrieval (HIER), and proteolytic-induced epitope retrieval (PIER). Selecting the right method is critical to get the best results for your application


HIER is the most commonly used method. It involves baking or microwaving the tissue sections in the presence of an antigen retrieval solution such as citrate or EDTA buffer. When using HIER, the choice of the buffer is important. Citrate, at pH 6, tends to unmask epitopes more conservatively than EDTA at pH 9. While EDTA can result in a more complete epitope unmasking, it can also increase background signal. Use serial sections to test which solution gives the best results, with proper controls to check for nonspecific staining.

PIER is most commonly performed with proteinase K, trypsin, or pepsin. PIER is much more aggressive than HIER and can destroy delicate morphological or antigenic features. This aggressiveness makes it unsuitable for delicate samples, but perfect for over-fixed, desiccated, or stubborn samples. PIER is generally not recommended for most sample types, as the intensity of the protein degradation is usually unnecessary to effectively unmask antigens.

Common Problems

Problems with the antigen retrieval step of IHC can result in a weak signal. Troubleshoot your IHC experiment with these tips:

  • Evaporation can cause the slide to dry out during AR; increase the volume of antigen retrieval solution in order to cover the slides more completely
  • Run a control experiment to determine the optimal amount of time for antigen retrieval. Use slides of the same tissue section and run the retrieval for 5, 10, 15, 20, 25 and 30 minutes before staining to evaluate optimal antigen retrieval time for the antibody being used.
  • Allow the slides to cool completely before handling. This allows the antigenic sites to re-form after being exposed to high temperatures.

More IHC Optimization Tips

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