Tomer Raju, Ye Li, Hsueh Brian, Deisseroth Karl
1] Department of Bioengineering, Stanford University, Stanford, California, USA. [2] Howard Hughes Medical Institute, Stanford University, Stanford, California, USA. [3] CNC Program, Stanford University, Stanford, California, USA.
1] Department of Bioengineering, Stanford University, Stanford, California, USA. [2] CNC Program, Stanford University, Stanford, California, USA.
Nat Protoc. 2014 Jul;9(7):1682-97. doi: 10.1038/nprot.2014.123. Epub 2014 Jun 19.
CLARITY is a method for chemical transformation of intact biological tissues into a hydrogel-tissue hybrid, which becomes amenable to interrogation with light and macromolecular labels while retaining fine structure and native biological molecules. This emerging accessibility of information from large intact samples has created both new opportunities and new challenges. Here we describe protocols spanning multiple dimensions of the CLARITY workflow, ranging from simple, reliable and efficient lipid removal without electrophoretic instrumentation (passive CLARITY) to optimized objectives and integration with light-sheet optics (CLARITY-optimized light-sheet microscopy (COLM)) for accelerating data collection from clarified samples by several orders of magnitude while maintaining or increasing quality and resolution. The entire protocol takes from 7-28 d to complete for an adult mouse brain, including hydrogel embedding, full lipid removal, whole-brain antibody staining (which, if needed, accounts for 7-10 of the days), and whole-brain high-resolution imaging; timing within this window depends on the choice of lipid removal options, on the size of the tissue, and on the number and type of immunostaining rounds performed. This protocol has been successfully applied to the study of adult mouse, adult zebrafish and adult human brains, and it may find many other applications in the structural and molecular analysis of large assembled biological systems.
CLARITY是一种将完整生物组织化学转化为水凝胶-组织杂交体的方法，该杂交体在保留精细结构和天然生物分子的同时，易于用光和大分子标记物进行检测。从大型完整样本中获取信息的这种新方法既带来了新机遇，也带来了新挑战。在此，我们描述了涵盖CLARITY工作流程多个维度的方案，从无需电泳仪器的简单、可靠且高效的脂质去除方法（被动CLARITY）到优化目标并与光片光学系统集成（CLARITY优化光片显微镜（COLM）），以便在保持或提高质量和分辨率的同时，将从澄清样本中收集数据的速度提高几个数量级。对于成年小鼠大脑，整个方案需要7至28天才能完成，包括水凝胶包埋、完全脂质去除、全脑抗体染色（如有需要，这占7至10天）以及全脑高分辨率成像；这个时间范围内的时长取决于脂质去除方法的选择、组织大小以及所进行的免疫染色轮次的数量和类型。该方案已成功应用于成年小鼠、成年斑马鱼和成年人类大脑的研究，并且可能在大型组装生物系统的结构和分子分析中找到许多其他应用。
