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Comparative analysis reveals Ce3D as optimal clearing method for in toto imaging of the mouse intestine
Author(s) -
Bossolani Gleison D. P.,
Pintelon Isabel,
Detrez Jan D.,
Buckinx Roeland,
Thys Sofie,
Zai Jacqueline Nelisis,
De Vos Winnok H.,
Timmermans JeanPierre
Publication year - 2019
Publication title -
neurogastroenterology and motility
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.489
H-Index - 105
eISSN - 1365-2982
pISSN - 1350-1925
DOI - 10.1111/nmo.13560
Subject(s) - clearing , reagent , small intestine , green fluorescent protein , biomedical engineering , biology , biophysics , chemistry , biochemistry , medicine , finance , gene , economics
Background 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. Aim The goal of this study was to identify an optimal clearing protocol for in toto imaging of mouse intestinal tissue. Methods 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 GFP/GFP and wild‐type mice. Following clearing, optical transparency, tissue morphology, green fluorescent protein (GFP) fluorescence retention, and compatibility with (immuno‐)labeling were analyzed. Key results 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. Conclusions Ce3D emerged as a most efficient protocol for enabling rapid full‐thickness 3D mapping of the mouse intestinal wall.