PH or Ionic Strength of Antigen Retrieval Solution: A Potential Role for Refolding during Heat Treatment

Yamashita, Shuji

doi:10.1002/9780470875612.ch17

Cited by 4 publications

(4 citation statements)

References 38 publications

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“…In their study, the precise amino acid composition of an epitope appears to be an important determinant of the precise conditions that are required for successful AR. Yamashita and others (Yamashita and Okada 2005b;Yamashita 2010) confirmed previous data that the pH and ionic strength of the retrieval solution are critical factors for AR efficiency in a mouse model, comparing SDS-PAGE gels of proteins and AR solutions at pH values ranging from 3 to 10.5.…”

Section: Mechanism Of Arsupporting

confidence: 70%

Antigen Retrieval Immunohistochemistry

Shi

Taylor

2011

J Histochem Cytochem.

230

105

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SummaryAs a review for the 20th anniversary of publishing the antigen retrieval (AR) technique in this journal, the authors intend briefly to summarize developments in AR-immunohistochemistry (IHC)-based research and diagnostics, with particular emphasis on current challenges and future research directions. Over the past 20 years, the efforts of many different investigators have coalesced in extending the AR approach to all areas of anatomic pathology diagnosis and research and further have led to AR-based protein extraction techniques and tissue-based proteomics. As a result, formalin-fixed paraffinembedded (FFPE) archival tissue collections are now seen as a literal treasure of materials for clinical and translational research to an extent unimaginable just two decades ago. Further research in AR-IHC is likely to focus on tissue proteomics, developing a more efficient protocol for protein extraction from FFPE tissue based on the AR principle, and combining the proteomics approach with AR-IHC to establish a practical, sophisticated platform for identifying and using biomarkers in

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Section: Mechanism Of Arsupporting

confidence: 70%

Antigen Retrieval Immunohistochemistry

Shi

Taylor

2011

J Histochem Cytochem.

230

105

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“…When FFPE specimens were autoclaved for 10 min at 120°C in 20 mM Tris-HCl buffer (pH 9.0), 50 mM citraconic anhydride aqueous solution (pH 7.4), or 10 mM citrate buffer (pH 6.0) containing 0, 50, 100, or 200 mM NaCl, all the antibodies showed the strongest immunostaining while the sections were autoclaved in the NaCl-free solutions. The staining intensity decreased as the NaCl concentration increased in all antibodies examined [24]. These results demonstrated that the ionic strength of the solution is a critical factor for HIAR and that a high concentration of salt inhibits the exposure of epitopes.…”

Section: Effect Of Ionic Strength Of Hiar Solutionmentioning

confidence: 63%

Antigen Retrieval for Light and Electron Microscopy

Yamashita¹

2020

Immunohistochemistry - The Ageless Biotechnology

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Heat-induced antigen retrieval (HIAR) method reported by Shi et al. in 1991 has greatly contributed not only to immunohistochemistry but also to studying gene expressions using archived formalin-fixed and paraffin-embedded (FFPE) specimens. Heating cleaves crosslinks (methylene bridges) in formaldehyde-fixed proteins and extends polypeptides to expose epitopes hidden in the inner portion of antigens or covered by adjacent macromolecules. In this chapter, the following topics are described to reconsider the concept of immunohistochemistry flexibly and to apply HIAR for further immunological studies using a variety of specimens: (1) antigen-antibody interactions in tissues; (2) mechanisms of chemical fixation with formaldehyde, glutaraldehyde, and osmium tetroxide; (3) unmasking of epitopes using HIAR for specimens fixed with chemical fixatives, including highly masked epitopes; (4) a standardized fixative for immunoelectron microscopy-based HIAR; (5) HIAR for conventionally processed electron microscopy specimens embedded in epoxy resins; and (6) effects of antibody diluents on immunohistochemistry.

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“…Antigen retrieval is an important step in the process of immunohistochemistry and is mainly based on heating induced protein modifications in order to release the antigen from methylene bridges formed between the proteins by formalin [22]. Some antigen retrieval procedures include pH modifications during heating in order to enhance the effect [23,24]. In case of alcohol-based fixatives, proteins undergo coagulation instead of forming of methylene bridges, and antigen retrieval developed for formalin-fixed tissues is ineffective resulting in lower IHC intensities [25].…”

Section: Discussionmentioning

confidence: 99%

“…All three cell lines were subjected to fixation in 10% neutral buffered formalin and CytoLyt ( Fig. 2a) for four different times (3,24,48, and 72 h). Formalin fixation of 24 h showed the highest H-scores and was considered the golden standard for further experiments.…”

Section: Variation In Fixatives and Fixation Timesmentioning

confidence: 99%

Influence of preanalytical variables on performance of delta-like protein 3 (DLL3) predictive immunohistochemistry

et al. 2020

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DLL3 might become a predictive immunohistochemical marker in small cell carcinoma of the lung (SCLC). We investigated the influence of pre-analytical handling of samples on the performance of DLL3 immunohistochemistry (IHC) using DLL3 SP347 ready to use assay (Ventana). DLL3 positive cell lines were subjected to different experimental conditions mimicking the pre-analytical variation in daily clinical practice. Formalin fixation of 24 h led to the most optimal results of DLL3 IHC. Longstanding fixation in Cytolyt, methanol-based fixative for cytology samples, but also decalcification using a mix of formic- and hydrochloracid resulted in decreased DLL3 staining. Postponed staining of blanc slides for 3 months also decreased DLL3 IHC. Postponed fixation of the SCLC cell lines did not influence the performance of DLL3 IHC, although this might be different in the tissues than in the cell lines. In conclusion, different pre-analytical variables decrease the performance of DLL3 IHC. These findings are essential for implementing novel predictive immunohistochemical biomarkers in daily pathology practice.

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PH or Ionic Strength of Antigen Retrieval Solution: A Potential Role for Refolding during Heat Treatment

Cited by 4 publications

References 38 publications

Antigen Retrieval Immunohistochemistry

Antigen Retrieval Immunohistochemistry

Antigen Retrieval for Light and Electron Microscopy

Influence of preanalytical variables on performance of delta-like protein 3 (DLL3) predictive immunohistochemistry

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