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Tissue Pretreatment: Heat-Induced Epitope Retrieval (HIER)

Heat-induced epitope retrieval (HIER) is required to expose many epitopes that have been altered or masked as a result of tissue fixation with formalin. Prior to the discovery of “antigen retrieval,” scientists were limited to the use of proteolytic enzymes that were not always compatible for exposing certain epitopes. As a result, weak or negative reactions with certain antibodies required the use of frozen tissue sections. The introduction of HIER techniques set the standard for current day IHC.
Doctors Fraenkel-Conrat and Olcott published studies in 1947 and 1948, that indicated cross-linkages between formalin fixation and tissue proteins could be disrupted by heating above 100° C or by strong alkaline treatment. It was not until the late 1980’s that Dr. Shi discovered the writings of Fraenkel-Conrat and Olcott. The first modern heat retrieval methodology using microwave energy and metal solutions was published by Shi, Key, and Kalra in 1991. This technology revolutionized IHC as we know it today.
Early problems associated with microwave energy were commonplace and included retrieval solution boil over, melting of lab ware, and the disruption of tissue due to the aggressive nature of the microwave process. This led investigators to explore alternate sources for heating the necessary solutions required for HIER protocols. In addition to microwaves being used for HIER protocols, autoclaves, table top pressure cookers, microwave pressure cookers, vegetable steamers, and high temperature water baths are also used. Pro and con issues are associated with all of the heating sources mentioned. However, with proper adherence to protocol and in the capable hands of a good immunohistochemist, proper results can be obtained with these various heat sources. Ultimately, the introduction of automated IHC staining platforms and online enzyme and heat retrieval techniques solved much of the aforementioned problems. Online HIER processes are consistent, more reproducible, less damaging to tissue sections, and a huge time saver. One major attribute to automation is standardization of protocol and reducing variability from technician to technician with manual HIER and IHC staining processes.
HIER solutions:
  • 10 mM sodium citrate, pH 6.0
  • 10 mM Tris, pH 9.5
  • 10 mM EDTA, pH 8.0
  • 10 mM Tris and 1-2 mM EDTA, pH 9.0
These solutions and variances of are commercially available in various concentrations of 10X, 20X, and 30X, as well as 1X ready to use. An Internet search can reveal formulas for the technologist who desires to prepare their own solutions.
The upper image shows HIER solution vessels placed in a high temperature water bath. Note the arrows indicating vinyl coated lead wire wrapped around the base of the plastic Coplin jars and the wire rack the stain dishes are sitting in. This will prevent the vessels from tipping over in a full water bath.
The lower image is an example of an analog bench top pressure cooker. Newer digital models are available from various vendors.