Journal article
An enhanced antigen-retrieval protocol for immunohistochemical staining of formalin-fixed, paraffin-embedded tissues
Methods in molecular biology (Clifton, N.J.), Vol.717, pp.101-110
2011
DOI: 10.1007/978-1-61779-024-9_6
PMID: 21370027
Abstract
Formalin is the most commonly used fixative for light microscopy because of its preservation of -morphological details. A major adverse effect of formalin fixation is formation of cross-linkages between epitopes (amino acid residues) and unrelated proteins by formaldehyde groups. The great majority of monoclonal and polyclonal antibodies used for immunohistochemical (IHC) staining of formalin-fixed, paraffin-embedded (FFPE) tissues necessitate unmasking antigens for antigen retrieval. There are currently two major antigen-retrieval procedures based on treatment of deparaffinized tissue sections with heat or, less commonly, with enzymatic digestion. The use of various antigen-retrieval solutions and heating sources does not allow standardization of IHC staining and minimalization of interlaboratory discrepancies. We developed a novel modified antigen-retrieval protocol for reversing the effect of -formalin fixation. The key feature of this protocol is treatment of deparaffinized tissue sections at reduced constant heat (97(o)C in a water bath) for 40 min in 25 mM Tris-HCl (pH 8.5), 1 mM EDTA, and 0.05% SDS (Tris-EDTA-SDS) buffer. Sections are then immunostained with primary and secondary antibodies conjugated with polymer-labeled Horse Radish Peroxidase. Compared to conventional antigen-retrieval procedures, this protocol more efficiently reverses the effect of formalin fixation of a wide variety of cellular antigens and in most instances decreases the use of primary antibody by 2-40 times, resulting in cost savings. Moreover, this protocol eliminates the need for using different antigen-retrieval methods in the laboratory, which reduces both time and labor for medical technologists.
Details
- Title: Subtitle
- An enhanced antigen-retrieval protocol for immunohistochemical staining of formalin-fixed, paraffin-embedded tissues
- Creators
- Sergei I Syrbu - Immunopathology Laboratory, Department of Pathology, The University of Iowa, Iowa City, IA, USA. sergei-syrbu@uiowa.eduMichael B Cohen
- Resource Type
- Journal article
- Publication Details
- Methods in molecular biology (Clifton, N.J.), Vol.717, pp.101-110
- Publisher
- United States
- DOI
- 10.1007/978-1-61779-024-9_6
- PMID
- 21370027
- eISBN
- 1617790249; 9781617790249
- ISSN
- 1064-3745
- eISSN
- 1940-6029
- Language
- English
- Date published
- 2011
- Academic Unit
- Pathology
- Record Identifier
- 9984046927602771
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