Antigen retrieval for IHC
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The process of sample fixation can lead to protein cross-linking, which masks antigens and can restrict antigen-antibody binding. Antigen retrieval enables an antibody to access the target protein within the tissue.
Masked epitopes can be recovered using either enzymatic/proteolytic antigen retrieval, or heat-induced antigen retrieval methods. In the enzymatic method, proteases such as proteinase K, trypsin, and pepsin are used. The heat-induced method uses heat (from either microwave, pressure cooker, steamer, waterbath, or autoclave) and a selection of buffers. The optimal antigen retrieval technique is dependent on the antigen, the tissue, the fixation method and/or primary antibody. Some antigens require a combination of heating and enzyme digestion.
To identify the best method for any particular antigen, we recommend testing two methods of heat-induced antigen retrieval (for example with citrate buffer pH 6 and Tris-EDTA pH 9) and one or two methods of enzymatic antigen retrieval (for example, with proteinase K and/or trypsin). Antigen retrieval may not be required for frozen sections where fixation is mild.
| HIER | PIER | |
| Advantages | Gentler epitope retrieval and more definable parameters | Useful for epitopes that are difficult to retrieve |
| pH | Citrate buffers of pH 6 are often used but high pH buffers have been shown to be widely applicable for many antibodies. Optimal pH must be determined experimentally. | Typically pH 7.4 |
| Temperature | Approximately 95˚C | Typically 37˚C |
| Incubation time | 10-20 minutes (commonly, 20 minutes) | 5-30 minutes (commonly, 10-15 minutes) |
| Buffer composition | Depends on pH required (pH is target-dependent, as shown in figure below). Popular buffers include sodium citrate, EDTA and Tris-EDTA | Neutral buffer solutions of enzymes such as pepsin, proteinase K or trypsin |
| Precautions | Heating using a microwave can result in unbalanced epitope retrieval due to uneven heating. Boiling can also lift tissue off of the slide. | Excessive enzymatic retrieval sometimes damages tissue morphology. |
The following figure illustrates the benefit of optimizing antigen retrieval to improve staining.
Effect of pH on heat-mediated antigen retrieval in human tissues From Emoto et al., (2005) Mechanisms of Heat-induced Antigen Retrieval: Does pH or Ionic Strength of the Solution Play a Role for Refolding Antigens? J Histochem Cytochem 53 (11):1311-21.
Buffers for heat-induced antigen retrieval
One step format antigen retrieval buffers contain detergents to allow deparaffinization and antigen retrieval to be performed in a single step to save time.
Antigen retrieval buffer | One step format | 100X | 10X |
Citrate buffer pH 6.0 | |||
EDTA buffer pH 8.0 | |||
Tris buffer pH 10.0 | |||
Tris-EDTA buffer pH 9.0 |
We also offer 10x Universal HIER antigen retrieval reagent ab208572 which is recommended for use with our PDL-1 antibody clone 28-8.
Buffers for enzymatic antigen retrieval
Buffer / enzyme formulation | Product |
HistoReveal (novel, rapid and gentle proteolytic treatment) | |
Pepsin | |
Proteinase K | |
Trypsin |
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