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IHC Antigen Retrieval

​Learn about the different methods of antigen retrieval.

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Aldehyde-based fixatives such as paraformaldehyde (PFA) or formalin act by creating cross-links between proteins in the tissue. In some cases, these cross-links mask epitopes, inhibiting antigen access by antibodies and resulting in weak or absent staining. Fortunately, this negative fixation can be reversed by a process called antigen retrieval. The type and method of antigen retrieval that works best depends on the tissue type and primary antibody. Our product-specific protocols state which method is best for each of our antibodies, validated using IHC.

Heat-induced Epitope Retrieval (HIER)

HIER is carried by heating the slides in a specific buffer for a certain period. Different heat sources like a microwave, pressure cooker, water bath, or a hot plate are normally used for this step.

If not specified by the antibody manufacturer, try HIER first with Tris-EDTA (pH 9) buffer. If this does not produce optimal retrieval, then it might be worth trying again with Citrate Buffer (pH 6). Determine the optimal antigen retrieval conditions for your samples by seeing which buffer produces the best staining. Further optimization can be achieved by varying the length of the incubation.

HIER acts by using thermal energy to break down cross-links that bind to the surrounding proteins or peptides. It is also thought to act through removing calcium ions from the site of protein cross-links.

General HIER Protocol

  • Heat the buffer to around 95°C (near boiling).
  • Apply the pre-heated buffer to the slides and incubate for 10-30 minutes.
  • Remove from the heat and let the slides cool in the buffer for a further 35 minutes.

Proteolytic-induced Epitope Retrieval (PIER)

Proteolytic-induced epitope retrieval (PIER) techniques result in a degradation of peptides to unmask the epitopes of interest. Trypsin and proteinase K are enzymes typically used in PIER, which act by breaking down protein cross-links, hence unmasking the hidden antigens and increasing staining intensity and specificity of the primary antibody.

General PIER Protocol

  • Prepare the trypsin and pre-heat to 37°C. Pipette the enzyme solution onto the section.
  • Place the slides in a humidified container and then into a 37°C incubator.
  • After 15 minutes, remove the slides from the incubator and transfer to a rack in a container with tap water. Rinse in running water for 3 minutes.

*Other commonly used enzymes are proteinase K, pepsin, and pronase.

Comparison of HIER vs PIER

  HIER PIER
Uses Most common, gentle epitope retrieval Good for difficult epitope recovery, can damage tissues
pH and Buffer Tris-EDTA pH 9 is the most used, citrate buffer pH 6 or other buffers at neutral pH can also be used pH 7.4; proteinase K, trypsin, pepsin, and pronase
Temperature 95°C 37°C
Incubation Time ~20 minutes ~10-15 minutes

*Optimal conditions should always be determined by each lab.