Ozawa Mina, Hattori Yujiro, Higo Shimpei, Otsuka Mai, Matsumoto Keisuke, Ozawa Hitoshi, Ishii Hirotaka
Department of Anatomy and Neurobiology, Graduate School of Medicine, Nippon Medical School, 1-1-5, Sendagi, Bunkyo-ku, Tokyo 113-8602, Japan.
School of Health Sciences, Bukkyo University, 7, Nishinokyo Higashitoganocho, Nakagyo-ku, Kyoto 604-8418, Japan.
Acta Histochem Cytochem. 2022 Oct 28;55(5):159-168. doi: 10.1267/ahc.22-00043. Epub 2022 Oct 25.
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.
尽管雌激素受体2（ESR2）具有生理意义，但缺乏经过充分验证的ESR2蛋白检测系统阻碍了ESR2研究的进展。因此，最近鉴定出一种特异性抗人ESR2单克隆抗体（PPZ0506）及其对小鼠和大鼠ESR2蛋白的特异性交叉反应性，这为开发适用于啮齿动物ESR2蛋白的免疫组织化学检测系统带来了新的契机。基于我们之前使用PPZ0506对大鼠ESR2免疫组织化学检测的优化，在本研究中，我们进一步旨在优化使用PPZ0506的小鼠对小鼠免疫组织化学检测。我们使用石蜡包埋的卵巢切片对几种染色条件进行评估，结果表明，强烈的热诱导抗原修复、适当的封闭和适当的抗体稀释对于优化小鼠对小鼠免疫组织化学是必要的。随后，我们应用优化后的免疫染色方法来确定小鼠ESR2蛋白在周围组织和脑区的表达谱。我们的分析显示，小鼠ESR2蛋白的分布比之前设想的更加局限。此外，将这些结果与使用PPZ0506在人类和大鼠中获得的结果进行比较，揭示了ESR2表达的种间差异。我们期望我们优化后的小鼠ESR2蛋白免疫组织化学染色方法将有助于研究人员解决有关ESR2表达的多条有争议的证据。
