Optimization of immunohistochemical detection of rat ESR2 proteins with well-validated monoclonal antibody PPZ0506

https://doi.org/10.1016/j.mce.2020.111145Get rights and content

Highlights

  • A well-validated anti-ESR2 antibody (PPZ0506) was recently identified.

  • Immunohistochemical staining of rat ESR2 proteins with the antibody was optimized.

  • The immunostaining required appropriate antigen retrieval and antibody dilution.

  • ESR2 distribution in rat tissues was examined under the optimized conditions.

  • The analysis revealed more localized ESR2 expression than previously assumed.

Abstract

Although there are few well-validated antibodies against ESR2 proteins, a recent validation assessment identified a specific monoclonal antibody against human ESR2 proteins (PPZ0506). Furthermore, our previous study confirmed its cross-reactivity and specificity against rodent ESR2 proteins, enabling the determination of true ESR2 distribution profiles in rodents. Therefore, we aimed to determine optimal conditions for ESR2 detection by PPZ0506 immunostaining and analyze ESR2 distribution in rats. We evaluated several staining conditions using paraffin-embedded and frozen ovary sections. Immunohistochemical staining with PPZ0506 antibody required strong antigen retrieval and appropriate antibody dilution. Subsequent immunohistochemical analysis in multiple tissues under optimized conditions revealed that rat ESR2 proteins are expressed in a more localized manner than previously assumed. Our results suggest that previous immunohistochemical studies using inadequately validated antibodies against ESR2 proteins overestimated their distribution profiles. We expect that optimized immunohistochemical detection with PPZ0506 antibody can help researchers solve several conflicting problems in ESR2 research.

Introduction

Estrogens play pivotal roles in both females and males in a wide variety of physiological and pathophysiological processes across multiple organs (Hamilton et al., 2017). Estrogens act through two forms of nuclear estrogen receptors (ERs), ESR1 and ESR2 (which are also known as ERα and ERβ, respectively; Dahlman-Wright et al., 2006). ESR1 is highly expressed in the reproductive organs and widely distributed in the nonreproductive organs (Hattori et al., 2015; Ishii and Sakuma, 2011; Ishii et al., 2013; Kobayashi et al., 2011). Although it is believed that ESR2 also exhibits wide distribution patterns, its detailed expression and localization profiles have not been determined because of a lack of appropriate tools to identify its distribution.

Research on ESR2 is one of the notorious cases in which studies have been distorted by the use of inadequately validated antibodies. Because of a paucity of well-validated antibodies against ESR2 proteins, multiple lines of controversial evidence regarding their distribution remain to be solved. Therefore, several efforts have been made to identify specific antibodies against ESR2 proteins (Nelson et al., 2017; Skliris et al., 2002; Snyder et al., 2010; Weitsman et al., 2006). Recently, Andersson et al. (2017) assessed the specificities of commercially available antibodies against human ESR2 proteins and determined that only one anti-human ESR2 monoclonal antibody (PPZ0506) displayed specific immunoreactivity to human ESR2 proteins and that it was applicable to immunohistochemical detection using human specimens. This discovery had a great impact on ESR2 research and resulted in several attempts to solve the controversial problems (Antonson et al., 2020; Hattori et al., 2020; Hawse et al., 2020; Ishii et al., 2019).

The PPZ0506 monoclonal antibody was developed using an N-terminal peptide (2–88 amino acids) of human ESR2 protein as an epitope. We have previously validated its cross-reactivity and specificity against rodent ESR2 proteins and determined its applicability in immunohistochemical detection of rat ESR2 proteins (Ishii et al., 2019). Our previous immunohistochemical analysis using PPZ0506 antibody demonstrated that the expression and localization profiles of rat ESR2 proteins were different from those of their human counterparts. Furthermore, we revealed that this species difference in the expression of ESR2 could be attributed to the distinct mRNA expression related to different profiles of alternative promoter usage between humans and rats. Despite the fact that our previous study indicated that ESR2 expression should be examined separately in humans and rats, conventional images of ESR2 expression were constructed by mixing the results obtained from human and rat tissues, based on the assumption that the histological expression profiles were nearly identical in humans and rats. These misconceptions are caused mainly by a paucity of reliable detection systems for ESR2. Therefore, to make progress in ESR2 research, precisely determining the expression and localization profiles of rat ESR2 proteins using a well-validated antibody is required.

In the present study, we first examine several staining conditions using the PPZ0506 antibody for optimization and reproducibility of immunohistochemical detection of rat ESR2 proteins. Subsequently, we apply the optimized methods to immunohistochemical analyses on the expression and localization of rat ESR2 proteins in the tissues, for comparison against previous studies that reported their immunoreactive signals using other inadequately validated antibodies against rat ESR2 proteins.

Section snippets

Animals

All animal experimental procedures in this study were approved by the Nippon Medical School Animal Care and Use Committee and conducted in compliance with the institutional guidelines. Wistar rats were purchased from Tokyo Laboratory Animals Science (Tokyo, Japan). The rats were housed in a temperature-controlled room (22°C–24 °C) under a 14-h light/10-h dark cycle. Standard diet and tap water were available ad libitum. Eight-to-10-week-old female rats at the diestrus stage and male rats were

Immunosignal detection

The immunohistochemical conditions were optimized using 5-μm-thick sections of paraformaldehyde-fixed and paraffin-embedded ovaries from diestrus female rats. The sections were deparaffinized, rehydrated, and then autoclaved for antigen retrieval in 10 mM citrate buffer (pH 6.0) at 121 °C for 10 min. In our preliminary immunohistochemical experiments, we used a conventional indirect immunodetection using a horseradish peroxidase–conjugated secondary antibody to detect immunoreactive signals of

Discussion

Despite the physiological importance of ESR2, a paucity of appropriate ESR2 detection systems has hindered progress in ESR2 research. Thus, discovery of a specific anti-human ESR2 monoclonal antibody (PPZ0506) and its applicability for immunodetection of rodent ESR2 proteins had great impacts on ESR2 research (Andersson et al., 2017; Ishii et al., 2019). In the present study, to further develop ESR2 research, we optimized the immunohistochemical detection system for rat ESR2 proteins.

Our

Funding

This work was supported by the Japan Society for the Promotion of Science KAKENHI Grants-in-Aid (grants 20K17544 [Y.H.], 18K06879 [H.I.], 18K16818 [M.K.], and 18K06860 [H.O.]).

CRediT authorship contribution statement

Yujiro Hattori: Methodology, Investigation, Visualization, Writing - review & editing, Funding acquisition. Hirotaka Ishii: Conceptualization, Methodology, Validation, Investigation, Visualization, Writing - original draft, Project administration, Funding acquisition. Shimpei Higo: Methodology, Validation, Visualization, Writing - review & editing. Mai Otsuka: Investigation. Moeko Kanaya: Methodology, Investigation, Funding acquisition. Keisuke Matsumoto: Investigation. Mina Ozawa:

Declaration of competing interest

The authors declare that they do not have any conflicts of interest to disclose.

Acknowledgments

We would like to thank Tatsuhiro Go for his helpful assistance with preliminary experiments to examine the optimal immunohistochemical conditions.

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    Present address: Moeko Kanaya; Department of Physiology, Division of Neurophysiology, School of Medicine, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan.

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