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. Author manuscript; available in PMC: 2013 Jun 12.
Published in final edited form as: Methods Mol Biol. 2013;980:29–42. doi: 10.1007/978-1-62703-287-2_3

Table 1.

Composition of buffers used for heat-induced epitope retrieval (HIER) and enzymatic antigen retrieval

Buffer Composition (1×) pH range Notes
Buffers used for heat-induced epitope retrieval (HIER)
Citrate buffer 10 mM Citric Acid (Sigma), 0.05% Tween 20 Adjust pH to 6.0 with 1 N NaOH, then add 0.5 ml of Tween 20, and mix well. (10× solution can be prepared and stored at 4°C for long-term use) Cross-links made by formalin or aldehyde fixers mask the antigen sites in tissue specimens leading false negative staining to some proteins, citrate buffers by breaking protein cross-links unmask the antigenic epitopes and enhance staining intensity of antibodies
EDTA buffer 1 mM EDTA (Sigma), 0.05% Tween 20 Adjust pH to 6.0 with 1 N NaOH, then add 0.5 ml of Tween 20, and mix well Works optimal for many antibodies; however, often results in high background staining (might be due to endogenous biotin revealed after this pretreatment). Buffer of choice for low-affinity antibodies and expression of antigen is quite low
Tris–EDTA buffer 10 mM Tris Base, 1 mM EDTA solution, 0.05% Tween 20 Adjust pH to 9.0 using 1 N NaOH, then add 0.5 ml of Tween 20, and mix well
Buffers used for enzymatic antigen retrieval (HIER)
Trypsin 0.05% Trypsin, 0.1% CaCl2 Adjust pH to 7.8 with 1 N NaOH Enzymes at high concentration destroy epitope and tissue morphology; therefore optimal enzyme concentration needs to be standardized for the antigen of interest. Standardize the time for enzymatic retrieval according to the antigen of choice
Apart from trypsin, pepsin and chymotrypsin should be checked for enzyme-based antigen retrieval