Retrieval is a technique practiced by immunohistochemistry (IHC) practitioners to expose tissue antigens masked by formalin fixation and make them abundantly available for the primary antibodies to bind.1,2 Formalin is the most frequently used fixative in the histology lab; fixing with 10 percent neutral buffered formalin is the standard fixation.2

In a nutshell, formalin fixation occurs by the reaction of formalin with the primary amines; the subsequent cross-linking of tissue proteins with formalin gives rise to formation of methylene bridges. Methylene bridges fix and preserve tissue antigens. Retrieval techniques are attempts to reverse formalin fixation by breaking methylene bridges and expose as many antigenic sites for primary antibodies to bind and, therefore, enhance the staining signals.

High Heat Retrieval (HIER)

HIER techniques are arbitrary and non-standardized practices with many variables.1-3 Deparaffinized tissue slides are immersed in varied buffers with different pHs and heated at boiling or slightly sub-boiling temperatures for 10-30 minutes. Slides then undergo a long cooling period before primary antibodies are applied. The selections for HIER heating time, buffers, pHs and heating devices are selected arbitrarily for each given primary antibody by both antibody vendors and IHC labs. It is important to note that the very high heat is the major factor in breaking the methylene bridges in HIER technique and the buffers used play a nominal role.

HIER, pH and Temperature Interplay

pH and temperature are inversely proportional. As the temperature rises, the pH drops. This phenomenon is the basis for the use of many HIER solutions with varied pHs. The two most commonly employed are citrate buffers pH 6 (acidic buffer) and high pH buffers pH 8-10 (alkaline buffer). The use of home-brewed solutions are also common and a minor variation on the basic theme.

Citrate buffer pH 6 was the original HIER buffer; high pH buffer arrived later. Citrate buffer pH 6 drops to pH 2-3 above temperature of 90o and higher, and such acidic pH is detrimental to the survival of many tissue antigens. The use of high pH buffer starting at pH 8-10 is an attempt to compensate for the drastic drop in pH associated with high heat in the HIER technique.

The high pH buffer is mainly used to provide a more neutral pH environment for tissue sections during the intense and pressurized heating period associated with HIER. Most tissue degradation and morphology damage in HIER are generated by extreme heat alone. EDTA added to high pH buffer has no effect on retrieval and no scientific merit. EDTA is a chelating agent; it may work well for bone tissues chelating the calcium but has no enhancing effect on retrieval or staining. EDTA containing retrieval buffers are not recommended for use with zinc formalin fixed tissues, as it chelates the zinc. Read more at the link below:
http://laboratory-manager.advanceweb.com/Article/IHC-Retrieval-Myths-Facts.aspx