Positive immunohistochemistry (IHC) controls are intended to detect problems in both immunostaining and heat-induced epitope retrieval (HIER). However, it is not known what features in a control are important for verifying HIER. Contrary to expectation, the fact that a tissue is formalin-fixed does not necessarily render it suitable in verifying proper HIER. Some tissue controls, for some immunostains, strongly stain even without HIER. Consequently, the control may verify the immunostain but provide little or no information regarding the HIER step. To sort this out, we used formalin-fixed peptide epitopes, a model that provides for precise definition of analyte concentration, epitope composition, and degree of fixation. Our data demonstrate that formalin fixation generates a variable level of protein epitope masking, depending on the epitope recognized by the primary antibody. Some epitopes are highly masked while others hardly at all. Furthermore, the ability of amino acids in the epitope to react with formaldehyde can, at least in part, account for this variability. Most important, we demonstrate the importance of selecting a positive control with a low or intermediate analyte concentration (relative to the immunostain's analytic sensitivity). High analyte concentrations can be insensitive in verifying the HIER step.

中文翻译：

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选择最佳IHC阳性阳性对照以验证抗原回收。

阳性免疫组织化学（IHC）对照旨在检测免疫染色和热诱导表位修复（HIER）方面的问题。但是，尚不知道控件中的哪些功能对于验证HIER很重要。与预期相反，组织被福尔马林固定的事实并不一定使其适合验证正确的HIER。对于某些免疫染色，即使没有HIER，某些组织对照也会强烈染色。因此，对照可以验证免疫染色，但是很少或没有提供有关HIER步骤的信息。为了解决这个问题，我们使用了福尔马林固定的肽表位，该模型提供了精确定义分析物浓度，表位组成和固定度的模型。我们的数据表明，福尔马林固定会产生可变水平的蛋白质表位掩蔽，取决于一抗识别的表位。一些表位被高度掩盖，而其他表位则几乎没有。此外，表位中的氨基酸与甲醛反应的能力可以至少部分地解释这种可变性。最重要的是，我们证明了选择低或中等分析物浓度（相对于免疫印迹分析灵敏度）的阳性对照的重要性。高浓度的分析物在验证HIER步骤时可能不敏感。我们证明选择低或中等分析物浓度（相对于免疫染色的分析灵敏度）的阳性对照的重要性。高浓度的分析物在验证HIER步骤时可能不敏感。我们证明选择低或中等分析物浓度（相对于免疫染色的分析灵敏度）的阳性对照的重要性。高浓度的分析物在验证HIER步骤时可能不敏感。