Mortazavi Farzad, Stankiewicz Alexander J, Zhdanova Irina V
Department of Anatomy and Neurobiology, Boston University School of Medicine, Boston, MA 02118, USA.
Department of Physics, University of Connecticut, Storrs, Connecticut 06269, USA.
Bio Protoc. 2019 Aug 5;9(15):e3321. doi: 10.21769/BioProtoc.3321.
Recently developed CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/Immunostaining/In situ-hybridization-compatible Tis-sue-hYdrogel) technique renders the tissue transparent by removing lipids in the tissue, while preserving and stabilizing the cellular and subcellular structures. This provides effective penetration of diverse labeling probes, from primary and secondary antibodies to complementary DNA and RNA strands. Followed by high-resolution 3D imaging of neuronal cells and their projections in thick sections, tissue blocks, whole brains, or whole animals, CLARITY allows for superior quantitative analysis of neuronal tissue. Here, we provide our detailed protocol for PACT (Passive Clarity Technique) in brain tissue of diverse species, including human, non-human primate, rodents, and zebrafish. We describe the six principal steps: (1) Tissue fixation and preparation, (2) Passive lipid removal, (3) Immuno-labeling, (4) Optical clearing, (5) Imaging, (6) 3D visualization and quantification.
最近开发的CLARITY（清除脂质交换丙烯酰胺杂交刚性成像/免疫染色/原位杂交兼容组织水凝胶）技术通过去除组织中的脂质使组织透明，同时保留并稳定细胞和亚细胞结构。这使得从一抗和二抗到互补DNA和RNA链等各种标记探针能够有效穿透。在对厚切片、组织块、全脑或全动物中的神经元细胞及其投射进行高分辨率3D成像之后，CLARITY允许对神经元组织进行卓越的定量分析。在这里，我们提供了针对不同物种（包括人类、非人类灵长类动物、啮齿动物和斑马鱼）脑组织的PACT（被动清除技术）详细方案。我们描述了六个主要步骤：（1）组织固定和制备，（2）被动脂质去除，（3）免疫标记，（4）光学清除，（5）成像，（6）3D可视化和定量。
