The impact of crosslinking and non-crosslinking fixatives on antigen retrieval and immunohistochemistry
The impact of crosslinking and non-crosslinking fixatives on antigen retrieval and immunohistochemistry
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DOI：
10.1016/j.nbt.2019.05.003
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发表时间：
2019-09-25
影响因子：
5.4
通讯作者：
Zatloukal, Kurt
中科院分区：
文献类型：
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作者：
Stumptner, Cornelia;Pabst, Daniela;Zatloukal, Kurt
在医学诊断和研究中，分析前因素会极大地影响分子分析的结果。这也适用于原位染色技术，例如免疫组织化学（MHC），其中不同类型的组织固定方法会导致蛋白质的不同修饰，从而会不同地影响抗体的检测。对于福尔马林固定石蜡包埋（FFPE）组织，应用抗原修复以逆转福尔马林的负面影响并重建免疫反应性。在IHC中使用的大多数抗体和协议进行了优化FFPE组织，但不是石蜡包埋的组织处理与其他固定剂，如非交联fixatives.We报告的结果从不同的预分析条件在IHC，免疫荧光和电子显微镜系统的研究。研究的参数是交联和非交联固定剂（比较福尔马林和PAXgene组织固定）对整个组织、亚细胞结构和细胞器以及超微结构的影响。生成的结果表明，抗原修复条件的微小变化可能对IHC结果产生重大影响，并且未经重新验证，针对交联固定剂优化的方案可能不适用于其他固定剂。对于每种抗体和固定方案，必须系统地测试关键抗原修复参数，例如具有不同pH值的缓冲液和微波处理持续时间。
Pre-analytical factors can greatly influence the outcome of molecular analyses in medical diagnostics and research. This also applies to in situ staining techniques such as immunohistochemistry (IHC), where different types of tissue fixation methods lead to different modifications of proteins and thus can affect differently the detection by antibodies. For formalin-fixed paraffin-embedded (FFPE) tissue, antigen retrieval is applied in order to reverse the negative effects of formalin and re-establish immunoreactivity. Most antibodies and protocols used in IHC are optimized for FFPE tissue, but not for paraffin-embedded tissue treated with other fixatives such as non-crosslinking fixatives.We report results from systematic studies on distinct pre-analytical conditions in IHC, immunofluorescence and electron microscopy. Parameters investigated are the impact of crosslinking and non-crosslinking fixatives (comparing formalin and PAXgene Tissue fixation) on whole tissue, subcellular structures and organelles, as well as on ultrastructure. The results generated show that minor changes in antigen retrieval conditions may have a major impact on IHC results and that protocols optimized for crosslinking fixatives may not be used for other fixatives without re-validation. Key antigen retrieval parameters such as buffers with different pH and duration of microwave treatment must be tested systematically for each antibody and fixation protocol.