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Despite the physiological significance of ESR2, a lack of well-validated detection systems for ESR2 proteins has hindered progress in ESR2 research. Thus, recent identification of a specific anti-human ESR2 monoclonal antibody (PPZ0506) and its specific cross-reactivity against mouse and rat ESR2 proteins heightened momenta toward development of appropriate immunohistochemical detection systems for rodent ESR2 proteins. Building upon our previous optimization of ESR2 immunohistochemical detection in rats using PPZ0506, in this study, we further aimed to optimize mouse-on-mouse immunohistochemical detection using PPZ0506. Our assessment of several staining conditions using paraffin-embedded ovary sections revealed that intense heat-induced antigen retrieval, appropriate blocking, and appropriate antibody dilutions were necessary for optimization of mouse-on-mouse immunohistochemistry. Subsequently, we applied the optimized immunostaining method to determine expression profiles of mouse ESR2 proteins in peripheral tissues and brain subregions. Our analyses revealed more localized distribution of mouse ESR2 proteins than previously assumed. Moreover, comparison of these results with those obtained in humans and rats using PPZ0506 revealed interspecies differences in ESR2 expression. We expect that our optimized methodology for immunohistochemical staining of mouse ESR2 proteins will help researchers to solve multiple lines of controversial evidence concerning ESR2 expression.

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Background: Research on ESR2, also known as estrogen receptor β (ERβ), is a notorious example of data distortion due to the use of inadequately validated antibodies. Although the absence of reliable specific antibodies against ESR2 has severely hindered the promotion of ESR2 research, a specific anti-human ESR2 monoclonal antibody (PPZ0506) was identified in 2017 [1]. Our previous study confirmed its cross-reactivity and specificity against rodent ESR2 proteins, enabling the elucidation of the true ESR2 distribution in rodents [2]. Objective: We aimed to determine the optimized conditions for immunohistochemical detection of rat ESR2 proteins using PPZ0506. <Method> Several staining conditions using paraffin-embedded and frozen ovary sections were evaluated, and the distribution of rat ESR2 proteins was analyzed under optimal conditions. Result: Immunohistochemical staining with PPZ0506 required appropriate antigen retrieval and 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 optimized immunohistochemical detection of rat ESR2 proteins, using a well-validated antibody, revealed their distribution in limited tissues and cell types. Conclusion: Our results suggest that previous immunohistochemical studies using inadequately validated antibodies against ESR2 proteins overestimated their distribution profiles. We expect that our optimized immunohistochemistry using the PPZ0506 antibody may solve conflicting problems in ESR2 research. References: 1. Andersson S, et al. Nat Commun 15;8:15840 (2017) 2. Ishii H, et al. Int J Mol Sci 20(24):6312 (2019)