Antibodies, A Laboratory Manual, Second Edition, Edited by Edward A. Greenfield



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Additional Protocol: Heat-Induced Epitope Retrieval

(Protocol summary only for purposes of this preview site)

Some antibodies bind poorly to antigens in cells and tissues that have been fixed and embedded in paraffin. This is because fixation sometimes masks the epitopes. Binding often improves following treatments that reverse the protein cross-links induced by formalin fixation and which unmask the epitopes. The most common technique consists of heating the tissue slides in an aqueous solution, often for up to 20 min, followed by cooling back to room temperature. The most common solutions for heat-induced epitope retrieval are citrate buffer (pH 6.0), EDTA buffer (pH 8.0), and Tris-EDTA buffer (pH 9.0), which may be prepared manually or purchased. The most common heating devices are standard, commercially available steam pressure cookers, vegetable steamers, and microwave ovens. Commercial, automated immunostainers often perform heat-mediated epitope retrieval as part of a programmable protocol.

Antibodies: A Laboratory Manual, Second edition
Antibodies: A Laboratory Manual, Second edition
Antibodies: A Laboratory Manual, Second edition

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