Ultra-fast and automated immunohistofluorescent multistaining using a microfluidic tissue processor
Ultra-fast and automated immunohistofluorescent multistaining using a microfluidic tissue processor
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DOI：
10.1038/s41598-019-41119-y
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发表时间：
2019-03-14
影响因子：
4.6
通讯作者：
Ciftlik, Ata Tuna
中科院分区：
文献类型：
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作者：
Cappi, Giulia;Dupouy, Diego Gabriel;Ciftlik, Ata Tuna
针对多个标记的组织切片的多重染色可以揭示肿瘤环境中复杂的相互作用。然而，迭代复用免疫染色的资源密集型和不切实际的长时间性质阻碍了它在日常工作中的实际实施，即使是在使用工作流自动化系统时。在这里，我们报告了一种全自动和超快速的多重染色，使用微流控组织处理器（MTP），每个标记只需20分钟，通过免疫荧光染色，利用市上可用的酪胺信号放大聚合物沉淀，通过马萝卜过氧化物酶（HRP）激活。报告的持续时间包括(i)免疫染色所需的整个流体交换和试剂孵育15分钟，（ii）热诱导去除所加抗体的5分钟。使用自动化MTP，我们演示了在84分钟内使用临床相关生物标志物进行4-plex自动多重染色，与最先进的微波治疗抗体去除完全一致。所提出的基于酶解酶的方法原则上可扩展到多重染色，既可容纳更多荧光通道的酪酰胺和多色显色染色。我们预计，我们的自动化多重染色的周期比现有的单免疫组织化学方法短，有可能在不干扰当前实验室工作流程的情况下进行常规多重染色，为在组织诊断中实施组学方法开辟前景。
Multistaining of a tissue section targeting multiple markers allows to reveal complex interplays in a tumor environment. However, the resource-intensive and impractically long nature of iterative multiplexed immunostainings prohibits its practical implementation in daily routine, even when using work-flow automation systems. Here, we report a fully automated and ultra-fast multistaining using a microfluidic tissue processor (MTP) in as short as 20 minutes per marker, by immunofluorescent staining employing commercially available tyramide signal amplification polymer precipitation by horse-radish peroxidase (HRP) activation. The reported duration includes (i) 15 minutes for the entire fluidic exchange and reagent incubation necessary for the immunostaining and (ii) 5 minutes for the heat-induced removal of the applied antibodies. Using the automated MTP, we demonstrated a 4-plex automated multistaining with clinically relevant biomarkers within 84 minutes, showing perfect agreement with the state-of-the-art microwave treatment antibody removal. The presented HRP-based method is in principle extendable to multistaining by both tyramides accommodating higher number of fluorescent channels and multi-color chromogenic staining. We anticipate that our automated multistaining with a turn-around time shorter than existing monoplex immunohistochemistry methods has the potential to enable multistaining in routine without disturbing the current laboratory workflow, opening perspectives for implementation of -omics approaches in tissue diagnostics.