Laboratory of Cell Biology and Histology, Department of Veterinary Sciences, University of Antwerp, Antwerp, Belgium.
Department of Morphological Sciences, State University of Maringá, Maringá, Brasil.
Neurogastroenterol Motil. 2019 May;31(5):e13560. doi: 10.1111/nmo.13560. Epub 2019 Feb 13.
The intestinal wall has a complex topographical architecture. The multi-layered network of the enteric nervous system and its intercellular interactions are difficult to map using traditional section-based or whole-mount histology. With the advent of optical clearing techniques, it has become feasible to visualize intact tissue and organs in 3D. However, as yet, a gap still needs to be filled in that no in-depth analysis has been performed yet on the potential of different clearing techniques for the small intestine.
The goal of this study was to identify an optimal clearing protocol for in toto imaging of mouse intestinal tissue.
Five aqueous-based clearing protocols (SeeDB2, CUBIC, ScaleS, Ce3D, and UbasM) and four organic reagent-based clearing protocols (3DISCO, iDISCO+, uDISCO, and Visikol ) were assessed in segments of small intestine from CX3CR1 and wild-type mice. Following clearing, optical transparency, tissue morphology, green fluorescent protein (GFP) fluorescence retention, and compatibility with (immuno-)labeling were analyzed.
All organic reagent-based clearing protocols-except for Visikol-rendered tissue highly transparent but led to substantial tissue shrinkage and deformation. Of the aqueous-based protocols, only Ce3D yielded full-thickness tissue transparency. In addition, Ce3D displayed excellent GFP retention and preservation of tissue morphology.
Ce3D emerged as a most efficient protocol for enabling rapid full-thickness 3D mapping of the mouse intestinal wall.
肠壁具有复杂的地形结构。肠神经系统的多层次网络及其细胞间相互作用很难使用传统的基于切片或全组织学进行映射。随着光学透明技术的出现，可视化完整组织和器官的 3D 结构成为可能。然而，目前仍然缺乏对不同透明技术在小肠中的潜在应用进行深入分析。
本研究旨在确定一种用于完整成像小鼠肠组织的最佳透明化方案。
评估了从小鼠 CX3CR1 和野生型肠道组织中分离的 5 种基于水的透明化方案（SeeDB2、CUBIC、ScaleS、Ce3D 和 UbasM）和 4 种基于有机试剂的透明化方案（3DISCO、iDISCO+、uDISCO 和 Visikol）。在透明化后，分析了光学透明度、组织形态、绿色荧光蛋白（GFP）荧光保留以及与（免疫）标记的兼容性。
除了 Visikol 之外，所有基于有机试剂的透明化方案都使组织高度透明，但导致组织严重收缩和变形。在基于水的方案中，只有 Ce3D 能使全层组织透明。此外，Ce3D 显示出优异的 GFP 保留和组织形态保存。
Ce3D 是一种最有效的方案，可用于快速实现小鼠肠壁的全层 3D 映射。
