Heat-induced antigen retrieval

Antigen antibody recognition is dependent on protein structure. A conformational change in a protein caused by formalin fixation may mask the epitope and thus affect the antigenicity of proteins in formalin-treated tissue (Montero 2003). The antigen retrieval leads to a renaturation or at least partial restoration of the protein structure, with re-establishment of the three-dimensional protein structure to something approaching its native condition (Shi et al. 1991). 

Antigen retrieval using a domestic vegetable steamer or a water bath Preheat steamer or water bath with a Coplin jar filled with an antigen retrieval solution of choice until temperature reaches 95°C 100°C. Immerse slides in the Coplin jar and place the lid loosely on the jar. Incubate for 30 60 min and turn off the steamer or water bath. Place the Coplin jar at room temperature and allow the slides to cool for 20 min. 

Before the beginning of the immunostaining protocol, slides should be put in a Coplin jar filled with an antigen retrieval solution of choice and heated in a commercial microwave oven operating at a frequency of 2.45 GHz and 600 W power setting. After two heating cycles of 5 min each, slides should be allowed to cool at room temperature and thoroughly washed in PBS. 

Target Retrieval Solutions from DAKO with pH 6.0 or pH 9.0 are well-suited for use on formalin-fixed, paraffin-embedded tissue sections mounted on glass slides. Compared with 0.01 M Citrate buffer, pH 6, the use of 0.001 M EDTA buffer, pH 9, significantly improves staining results for many antigens, preserves the morphology better and is especially useful in combination with the Dako EnVision visualization systems. 

Tris buffered saline (TBS) (stock solution x 10 concentrated) 

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Yamashita S, Okada Y. Mechanisms of heat-induced antigen retrieval analyses in vitro employing SDS-PAGE and immunohistochemistry. J. Histochem. Cytochem. 2005 53 13-21. 

Yamashita S. Heat-induced antigen retrieval mechanisms and application to histochemistry. Prog. Histochem. Cytochem. 2007 41 141-200. 

BoenischT. Effect of heat-induced antigen retrieval following inconsistent formalin fixation. Appl. Immunohistochem. Mol. Morphol. 2005 13 283-286. 

HEAT-INDUCED ANTIGEN RETRIEVAL (HIAR) METHODS ... 

In 1991 Shi et al. published their seminal observation that high-temperature incubation of formalin-fixed, paraffin-embedded (FFPE) tissue sections in buffers for short periods led to improved immunohistochemical staining.1 However, more than 15 years later, heat-induced antigen retrieval (AR) remains largely an empirical procedure, requiring the optimization of several critical parameters by trial and error.2,3 Further improvements in AR will require an in-depth understanding of the chemistry of formaldehyde fixation and the molecular mechanism(s) underlying the AR method. 

Kakimoto K, Takekoshi S, Miyajima K, et al. Hypothesis for the mechanism for heat-induced antigen retrieval occurring on fresh frozen sections without formalin-fixation in immunohistochemistry. J. Mol. Histol. 2008 39 389-399. 

Boenisch T. Heat-induced antigen retrieval what are we retrieving [Review]. J. Histochem. Cytochem. 2006 54 961-964. 

Brorson SH. Heat-induced antigen retrieval of epoxy sections for electron microscopy. Histol. Histopathol. 2001 16 923-930. 

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