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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.
|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
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
HistoReveal (novel, rapid and gentle proteolytic treatment)