IHC guide v3 952x200px

Antigen retrieval for IHC

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. 

AdvantagesGentler epitope retrieval and more definable parametersUseful for epitopes that are difficult to retrieve
pHCitrate 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
TemperatureApproximately 95˚CTypically 37˚C
Incubation time10-20 minutes (commonly, 20 minutes)5-30 minutes (commonly, 10-15 minutes)
Buffer compositionDepends on pH required (pH is target-dependent, as shown in figure below). Popular buffers include sodium citrate, EDTA and Tris-EDTANeutral buffer solutions of enzymes such as pepsin, proteinase K or trypsin
PrecautionsHeating 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)




Proteinase K




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