Epitope retrieval

Gown AM. Unmasking the mysteries of antigen or epitope retrieval and formalin fixation. Am. J. Clin. Pathol. 2004 121 172-174. 

Rahimi F, Shepherd CE, Halliday GM, et al. Antigen-epitope retrieval to facilitate proteomic analysis of formalin-fixed archival brain tissue. Anal. Chem. 2006 78 7216-7221. 

Miller RT, Swanson PE, Wick MR. Fixation and epitope retrieval in diagnostic immunohistochemistry a concise review with practical considerations. Appl. Immunohistochem. Mol. Morphol. 2000 8 228-235. 

Man, Y-g. and Tavassoli, F. A. (1996) A simple epitope retrieval method without the use of microwave oven or enzyme digestion. Appl. Immunohistochem. 4, 139-141. 

Despite some success with the above pretreatments, the development of wet heat-induced epitope retrieval (HIER) procedures has been the critical... 

Leong, A. S. -Y., Milios, J., and Leong, F. J. (1996) Epitope retrieval with micro-waves. A comparison of citrate buffer and EDTA with three commercial retrieval solutions. Appl. Immunohistochem. 4, 201-207. 

It should also be noted that denaturing agents make cells permeable, facilitating antibody penetration. Moreover, these agents are used usually in combination with microwave heating. Therefore, the role of these agents in epitope retrieval needs to be explained in the context of their role in cell permeabilization as well as of the influence of elevated temperatures. 

Type of microwave oven and heating parameters, other heating sources, or other epitope retrieval treatments such as enzyme digestion and ultrasound... 

The following three levels of heating durations and three pH levels of the epitope retrieval fluid (sodium citrate buffer and Tris-HCl buffer) have been recommended to determine the optimal protocol for the retrieval of an epitope (Shi et al., 1996a). 

Is it practical to develop a universal epitope retrieval method The answer is no, as the optimal retrieval of each type of epitope requires very specific processing conditions such as fixation, retrieval fluid, and unmasking treatments, including heating, enzymatic digestion, and ultrasound. In terms of preservation and masking, each type of epitope is affected differently by the fixative used, and by its concentration, pH, and the temperature and duration of fixation. As long as different fixatives are used in different laboratories,... 

Epitope retrieval depends upon epitope preservation and hence on the effects of fixation or chemical fixation. In some cases, the fixation history of archival tissues is not known, making the rational choice of epitope retrieval methods problematic. Another problem is that similar antibodies obtained from different sources may vary in their sensitivity and specificity. 

That heat is not the only method to unmask epitopes is exemplified by enzyme digestion or detergent treatment. The exact mechanism responsible for epitope retrieval with ultrasound is not clear, although intense heat is produced for an exceedingly short duration. It is known, however, that ultrasound and/or heat decreases the amount of negative charges on the cell surface (Joshi et al., 1983 Adler et al., 1988). Mechanical vibrations of molecules caused by ultrasound and heat are thought to unfold the protein molecule and to expose the epitopes. 

Because heating in water alone did not improve immunostaining, it is concluded that epitope retrieval is mediated not only by heat and rehydration but also by the presence of a chelating agent such as EDTA. However, caution is warranted in using EDTA, which may adversely affect cell morphology because it is a strong oxidant. 

The effects of microwave heating on the tissue sections that result in epitope retrieval are exceedingly complex. A full understanding of the actions of microwaves at the molecular level to facilitate epitope retrieval is lacking. At least two mechanisms need to be considered heat and kinetic energy of the oscillating electromagnetic field. Both possibilities are discussed below. 

The most commonly accepted point of view is that heat is responsible for unmasking the epitopes. In fact, Battifora (1996) has introduced the phrase heat-induced epitope retrieval (HIER). Heating at KXPC is a powerful treatment that can unmask hidden, buried, or crosslinked epitopes. Heat can be provided not only by a microwave oven, but also by an autoclave, a pressure cooker, steam, or a hot plate. A consensus on which method of heating is most effective in the retrieval of all types of epitopes is lacking. Therefore, some... 

The following hydrated autoclave method can be employed for immunohistochemical detection of molecules in both cultured cell and tissue specimens. The method was used, for example, to localize androgen receptor in cultured LNCaP cells (derived from prostatic carcinoma metastasized to lymph node) and biopsy specimens from patients with prostatic carcinoma (Ehara et al 1996). After being removed from the culture medium, the cells on plastic cover slips are fixed with 10% formalin for 10 min at 20°C. Tissue specimens are fixed for 1-2 days and embedded in paraffin. Sections (5 pan) are cut, mounted on glass slides, and heated in an oven for 1 hr at 42°C to promote adherence to the slide. After deparaffmizing and rehydration, the sections are subjected to epitope retrieval treatment as follows. 

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