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Published on: July 19, 2024
An enhanced antigen-retrieval protocol for immunohistochemical staining of formalin-fixed, paraffin-embedded tissues
Sergei I Syrbu1, Michael B Cohen
1Immunopathology Laboratory, Department of Pathology, The University of Iowa, Iowa City, IA, USA. sergei-syrbu@uiowa.edu
Insights
A new antigen retrieval protocol effectively reverses formalin fixation in tissues, improving immunohistochemical staining. This method reduces antibody use and laboratory discrepancies, saving time and costs.
Area of Science:
- Histopathology
- Immunohistochemistry
- Biochemistry
Background:
- Formalin fixation preserves tissue morphology but causes protein cross-linking, hindering antibody binding for immunohistochemistry (IHC).
- Current antigen retrieval methods, using heat or enzymes, lack standardization and contribute to interlaboratory variability.
- Antigen retrieval is crucial for unmasking epitopes in formalin-fixed, paraffin-embedded (FFPE) tissues for effective IHC staining.
Purpose of the Study:
- To develop a novel, standardized antigen retrieval protocol to overcome formalin fixation-induced epitope masking.
- To improve the efficiency and reliability of immunohistochemical staining in FFPE tissues.
- To reduce primary antibody consumption and associated costs in IHC.
Main Methods:
- A modified antigen retrieval protocol involving deparaffinized tissue sections treated at 97°C for 40 minutes in a Tris-EDTA-SDS buffer.
- Subsequent immunostaining using polymer-labeled Horse Radish Peroxidase conjugated primary and secondary antibodies.
- Comparison of the novel protocol against conventional heat- and enzyme-based antigen retrieval methods.
Main Results:
- The novel protocol efficiently reverses formalin fixation for a broad range of cellular antigens.
- Significant reduction in primary antibody usage (2-40 fold) was observed compared to conventional methods.
- The protocol demonstrated potential for cost savings and reduced interlaboratory discrepancies in IHC staining.
Conclusions:
- The developed modified antigen retrieval protocol offers a standardized and efficient method for IHC staining of FFPE tissues.
- This protocol effectively unmasks antigens, reduces antibody consumption, and minimizes variability, leading to cost and labor savings.
- This approach simplifies IHC workflows by eliminating the need for diverse retrieval methods in the laboratory.
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.
