Epitope Retrieval

IHC Tissue Microarrays

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Sample Preparation

Sample Fixation

Blocking Non-Specific Binding

Primary Antibody Selection

IHC Detection

Epitope Retrieval

Why is antigen retrieval necessary? The impaired ability of antibodies to access epitope in fixed tissue can impact IHC staining. Protein fixation creates crosslinks that mask tissue antigen and limit antibody-epitope binding. In addition to changes in epitope conformation and electrostatic charge, masking is the result of crosslinks created between amino acids within the target antigen and between proteins surrounding the target antigen.

What is antigen retrieval and how does it work? Antigen retrieval refers to any method which restores antigenicity and enhances antibody-epitope binding by reversing protein crosslinks in tissue sections.

When should I run an antigen retrieval step? Antigen retrieval is recommended for most tissue antigens following deparaffinization and rehydration of formalin-fixed tissues in the immunohistochemistry staining protocol.

Is antigen retrieval necessary on frozen tissue sections? Antigen retrieval on frozen tissue is not recommended. The retrieval process can be too harsh and damage the tissue. However, it is often recommended to restore antigenicity in formalin-fixed tissues.

Is antigen retrieval always required on formalin-fixed paraffin-embedded tissues? Not all IHC experiments require a retrieval step. The fixation method and length, in addition to the type of tissue, target antigen, and antibody determine the need for antigen retrieval. For instance, without an antigen retrieval step, a polyclonal antibody may enhance detection of antigen compared to a monoclonal due to its ability to bind multiple epitopes. A change in pH or cation concentration of antibody diluent or a simple change in the incubation conditions of primary antibody can also improve antibody affinity for its epitope without the need for a formal antigen retrieval step. However, antigen retrieval is recommended for most formalin-fixed paraffin-embedded tissues.

What is the difference between heat-induced epitope retrieval and proteolytic-induced epitope retrieval? One of two antigen retrieval methods is recommended on most formalin-fixed paraffin-embedded tissue sections. Proteolytic-Induced Epitope Retrieval (PIER) is an enzymatic method of antigen retrieval which relies on enzymes such as proteinase K, trypsin, or pepsin to unmask antigen. Heat-Induced Epitope Retrieval (HIER) utilizes heat to promote epitope availability. Because ideal retrieval conditions are influenced by the tissue type and fixation method, optimization of the retrieval protocol for each antigen is highly recommended.


What is it?

How does it work?

Heat-Induced Epitope Retrieval The use of heat to retrieve antigen and restore antigenicity Heat causes crosslinked protein to unfold.
Proteolytic-Induced Epitope Retrieval The use of enzymes to retrieve antigen and restore antigenicity Enzymes degrade protein crosslinks


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Optimization of Antigen Retrieval

The identification of optimal conditions for antigen retrieval requires initial testing. Once optimized, the effects of retrieval can have a significant impact on signal strength and target protein identification. Due to inconsistency in the strength of heating apparatuses employed in HIER protocols, the ideal time, temperature, and pH for each antigen should be optimized to reduce variability. For example, 5-10 minutes at 92-95 °C in a water bath may equate to 1-5 minutes at 120 °C in a pressure cooker. When antigen retrieval methods are used prior to primary antibody incubation, staining artifacts should be considered. To ensure specific staining and eliminate the possibility of artifacts, appropriate controls should be run.


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Optimization of antigen retrieval

Antigen Retrieval Improves Detection of p27. IHC images show the detection of p27 in paraffin-embedded human prostate cancer sections following incubation of tissue for 10 minutes at 95 °C in the specified antigen retrieval solution. Compared to no HIER treatment, p27 detection was enhanced following incubation in neutral (pH 7.0) and basic (pH 9.5,) but not acidic (pH 5.0) antigen retrieval solution. P27 was detected using anti-human/mouse/rat p27 monoclonal antibody (Catalog #MAB22561; brown). Image from R&D Systems: Antigen Retrieval Methods.