Antigen Retrieval Immunohistochemistry

Shi, Shan‐Rong; Shi, Yan; Taylor, Clive R.

doi:10.1369/jhc.2010.957191

Cited by 226 publications

(106 citation statements)

References 187 publications

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“…Tumor tissues were fixed, and tissue sections were embedded in paraffin and subjected to antigen retrieval in a microwave in Tris-HCl-EDTA buffer (pH 8.0) as described previously (27). Sections were incubated with anti-CD4 and anti-CD25 antibodies (Thermo Fisher Scientific) and subsequently with horseradish peroxidase (HRP)-conjugated secondary antibodies and were counterstained with hematoxylin.…”

Section: Methodsmentioning

confidence: 99%

Superoxide-Generating Nox5α Is Functionally Required for the Human T-Cell Leukemia Virus Type 1-Induced Cell Transformation Phenotype

Shigemura

Shiohara

Kato

et al. 2015

J Virol

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Human T-cell leukemia virus type 1 (HTLV-1) is associated with adult T-cell leukemia (ATL) and transforms T cells in vitro.To our knowledge, the functional role of reactive oxygen species (ROS)-generating NADPH oxidase 5 (Nox5) in HTLV-1 transformation remains undefined. Here, we found that Nox5␣ expression was upregulated in 88% of 17 ATL patient samples but not in normal peripheral blood T cells. Upregulation of the Nox5␣ variant was transcriptionally sustained by the constitutive Janus family tyrosine kinase (Jak)-STAT5 signaling pathway in interleukin-2 (IL-2)-independent HTLV-1-transformed cell lines, including MT1 and MT2, whereas it was transiently induced by the IL-2-triggered Jak-STAT5 axis in uninfected T cells. A Nox inhibitor, diphenylene iodonium, and antioxidants such as N-acetyl cysteine blocked proliferation of MT1 and MT2 cells. Ablation of Nox5␣ by small interfering RNAs abrogated ROS production, inhibited cellular activities, including proliferation, migration, and survival, and suppressed tumorigenicity in immunodeficient NOG mice. The findings suggest that Nox5␣ is a key molecule for redox-signal-mediated maintenance of the HTLV-1 transformation phenotype and could be a potential molecular target for therapeutic intervention in cancer development. IMPORTANCEHTLV-1 is the first human oncogenic retrovirus shown to be associated with ATL. Despite the extensive study over the years, the mechanism underlying HTLV-1-induced cell transformation is not fully understood. In this study, we addressed the expression and function of ROS-generating Nox family genes in HTLV-1-transformed cells. Our report provides the first evidence that the upregulated expression of Nox5␣ is associated with the pathological state of ATL peripheral blood mononuclear cells and that Nox5␣ is an integral component of the Jak-STAT5 signaling pathway in HTLV-1-transformed T cells. Nox5␣-derived ROS are critically involved in the regulation of cellular activities, including proliferation, migration, survival, and tumorigenicity, in HTLV-1-transformed cells. These results indicate that Nox5␣-derived ROS are functionally required for maintenance of the HTLV-1 transformation phenotype. The finding provides new insight into the redox-dependent mechanism of HTLV-1 transformation and raises an intriguing possibility that Nox5␣ serves as a potential molecular target to treat HTLV-1-related leukemia.H uman T-cell leukemia virus type 1 (HTLV-1) is the first human oncogenic retrovirus shown to be etiologically associated with adult T-cell leukemia (ATL) (1, 2). ATL has a poor prognosis because of its resistance to conventional chemotherapy, and no effective therapy is currently available for ATL. HTLV-1 infects and transforms human peripheral blood T cells in vitro (3,4), but the precise mechanism of HTLV-1 transformation of T cells and the development of ATL after HTLV-1 infection are not fully understood. A HTLV-1 genome pX region-encoded protein, Tax, is thought to play a central role in activation, proliferation, and transfor...

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Section: Methodsmentioning

confidence: 99%

Superoxide-Generating Nox5α Is Functionally Required for the Human T-Cell Leukemia Virus Type 1-Induced Cell Transformation Phenotype

Shigemura

Shiohara

Kato

et al. 2015

J Virol

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“…In such situations, heat-or proteolysis-induced epitope retrieval can be applied to improve the signal [119,120].…”

Section: Immunohistochemistry With Tissue Slicesmentioning

confidence: 99%

Immunodiagnostics and immunosensor design (IUPAC Technical Report)

Gubala

Klein²,

Templeton

et al. 2014

Pure and Applied Chemistry

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This work compiles information on the principles of diagnostic immunochemical methods and the recent advances in this field. It presents an overview of modern techniques for the production of diagnostic antibodies, their modification with the aim of improving their diagnostic potency, the different types of immunochemical detection systems, and the increasing diagnostic applications for human health that include specific disease markers, individualized diagnosis of cancer subtypes, therapeutic and addictive drugs, food residues, and environmental contaminants. A special focus lies in novel developments of immunosensor techniques, promising approaches to miniaturized detection units and the associated microfluidic systems. The trends towards high-throughput systems, multiplexed analysis, and miniaturization of the diagnostic tools are discussed. It is also made evident that progress in the last few years has largely relied on novel chemical approaches.

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“…Similar challenges are encountered for HIC of FFPE samples. Over time, many different protocols have been developed to unmask antigen epitopes [24,[51][52][53][54][55][56][57][58][59][60]. These so-called AR processes are still not well understood in terms of the associated molecular changes.…”

Section: In-source Decay Approachmentioning

confidence: 99%

“…The most reasonable hypothesis is that AR allows changes in the conformation of cross-linked proteins and thus facilitates access to sites within the proteins. To date, many different AR protocols have been published [24,[51][52][53][54][55][56][57][58][59][60]. However, according to the nature of the tissue used and the protein (antigen) under consideration, some protocols work better than others which are not yet well understood.…”

Section: In-source Decay Approachmentioning

confidence: 99%

Multivariate analyses for biomarkers hunting and validation through on-tissue bottom-up or in-source decay in MALDI-MSI: application to prostate cancer

Bonnel

Longuespée

Franck³

et al. 2011

Anal Bioanal Chem

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The large amount of data generated using matrixassisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI) poses a challenge for data analysis. In fact, generally about 1.10 8 -1.10 9 values (m/z, I) are stored after a single MALDI-MSI experiment. This imposes processing techniques using dedicated informatics tools to be used since manual data interpretation is excluded. This work proposes and summarizes an approach that utilizes a multivariable analysis of MSI data. The multivariate analysis, such as principal component analysis-symbolic discriminant analysis, can remove and highlight specific m/z from the spectra in a specific region of interest. This approach facilitates data processing and provides better reproducibility, and thus, broadband acquisition for MALDI-MSI should be considered an effective tool to highlight biomarkers of interest. Additionally, we demonstrate the importance of the hierarchical classification of biomarkers by analyzing studies of clusters obtained either from digested or undigested tissues and using bottom-up and in-source decay strategies for in-tissue protein identification. This provides the possibility for the rapid identification of specific markers from different histological samples and their direct localization in tissues. We present an example from a prostate cancer study using formalinfixed paraffin-embedded tissue.

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Antigen Retrieval Immunohistochemistry

Cited by 226 publications

References 187 publications

Superoxide-Generating Nox5α Is Functionally Required for the Human T-Cell Leukemia Virus Type 1-Induced Cell Transformation Phenotype

Superoxide-Generating Nox5α Is Functionally Required for the Human T-Cell Leukemia Virus Type 1-Induced Cell Transformation Phenotype

Immunodiagnostics and immunosensor design (IUPAC Technical Report)

Multivariate analyses for biomarkers hunting and validation through on-tissue bottom-up or in-source decay in MALDI-MSI: application to prostate cancer

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