Urea-based amino sugar agent clears murine liver and preserves protein fluorescence and lipophilic dyes.
Urea-based amino sugar agent clears murine liver and preserves protein fluorescence and lipophilic dyes.
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DOI：
10.2144/btn-2020-0063
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发表时间：
2021-03
期刊：
影响因子：
2.7
通讯作者：
Wang KS
中科院分区：
文献类型：
--
作者：
Hough M;Fenlon M;Glazier A;Short C;Fernandez GE;Xu J;Mahdi E;Asahina K;Wang KS
五个已建立的清除协议进行了比较，修改和简化的方法，以确定一个最佳的清除试剂的三维可视化荧光团在小鼠肝脏，一个具有挑战性的器官清除。我们报告了通过修改仅使用Ub-1（一种基于尿素的氨基糖试剂）的既定方案（UbasM）成功清除整个肝叶的简单方案，仅需要24小时的处理时间。单独使用Ub-1时，我们观察到在三维沿着中充分保留的肝组织结构，以及来自内源性蛋白质报告子和亲脂性示踪染料的荧光团发射的优异保留。这种流线型技术可用于3D细胞谱系追踪和基于荧光探针的报告基因表达，以比较各种实验条件。本研究提出了一种简化的方案，用于在仅24小时内使用一种简单的基于尿素的氨基糖溶液和单次孵育来光学地清除小鼠肝脏组织。该方法保留了天然空间形态背景下转基因表达的蛋白和亲脂性示踪染料的荧光。
Five established clearing protocols were compared with a modified and simplified method to determine an optimal clearing reagent for three-dimensionally visualizing fluorophores in the murine liver, a challenging organ to clear. We report successful clearing of whole liver lobes by modification of an established protocol (UbasM) using only Ub-1, a urea-based amino sugar reagent, in a simpler protocol that requires only a 24-h processing time. With Ub-1 alone, we observed sufficiently preserved liver tissue structure in three dimensions along with excellent preservation of fluorophore emissions from endogenous protein reporters and lipophilic tracer dyes. This streamlined technique can be used for 3D cell lineage tracing and fluoroprobe-based reporter gene expression to compare various experimental conditions. This study presents a simplified protocol for optically clearing murine liver tissue in only 24 h using one simple urea-based amino sugar solution and a single incubation. This method preserves fluorescence of transgenically expressed proteins and lipophilic tracer dyes within the context of native spatial morphology.