用经过充分验证的单克隆抗体 PPZ0506 优化免疫组化检测大鼠 ESR2 蛋白。

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

机构信息

Department of Anatomy and Neurobiology, Graduate School of Medicine, Nippon Medical School, 1-1-5, Sendagi, Bunkyo-ku, Tokyo, 113-8602, Japan.

出版信息

Mol Cell Endocrinol. 2021 Mar 1;523:111145. doi: 10.1016/j.mce.2020.111145. Epub 2021 Jan 2.

DOI:10.1016/j.mce.2020.111145

PMID:33400952

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.

摘要

虽然针对 ESR2 蛋白的抗体验证较少，但最近的验证评估确定了一种针对人 ESR2 蛋白的特异性单克隆抗体（PPZ0506）。此外，我们之前的研究证实了它对啮齿动物 ESR2 蛋白的交叉反应性和特异性，从而能够确定啮齿动物中真正的 ESR2 分布模式。因此，我们旨在通过 PPZ0506 免疫染色确定 ESR2 检测的最佳条件，并分析大鼠中 ESR2 的分布。我们使用石蜡包埋和冷冻卵巢切片评估了几种染色条件。PPZ0506 抗体的免疫组织化学染色需要强烈的抗原修复和适当的抗体稀释。在优化条件下对多种组织进行的后续免疫组织化学分析表明，大鼠 ESR2 蛋白的表达比以前假设的更局限。我们的结果表明，以前使用针对 ESR2 蛋白的验证不足的抗体进行的免疫组织化学研究高估了它们的分布模式。我们期望使用 PPZ0506 抗体进行优化的免疫组织化学检测可以帮助研究人员解决 ESR2 研究中的几个相互矛盾的问题。