Optimization of immunohistochemical detection of rat ESR2 proteins with well-validated monoclonal antibody PPZ0506
Optimization of immunohistochemical detection of rat ESR2 proteins with well-validated monoclonal antibody PPZ0506
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DOI：
10.1016/j.mce.2020.111145
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发表时间：
2021-01-11
影响因子：
4.1
通讯作者：
Ozawa, Hitoshi
中科院分区：
文献类型：
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作者：
Hattori, Yujiro;Ishii, Hirotaka;Ozawa, Hitoshi
尽管针对ESR2蛋白的抗体很少经过充分验证，但最近的验证评估鉴定出了针对人ESR2蛋白的特异性单克隆抗体（PPZ 0506）。此外，我们之前的研究证实了其对啮齿动物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实的ESR2分布特征。因此，我们的目的是确定通过PPZ 0506免疫染色检测ESR2的最佳条件并分析ESR2在大鼠体内的分布。我们使用石蜡包埋和冷冻卵巢切片评估了几种染色条件。用PPZ 0506抗体进行免疫组织化学染色需要强抗原修复和适当的抗体稀释。随后在优化条件下在多个组织中进行的免疫组织化学分析显示，大鼠ESR2蛋白的表达比先前假设的更局部化。我们的研究结果表明，以前的免疫组织化学研究使用不充分验证的抗体对ESR2蛋白高估了他们的分布概况。我们希望PPZ0506抗体的优化免疫组化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。
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.