Comparison of different clearing protocols for in toto three‐dimensional microscopic imaging of the intestinal wall
Authors: Bossolani Gleison D.P. , Detrez Jan R. , Zanoni Jacqueline Nelis , Adriaensen Dirk , De Vos Winnok H. , Buckinx Roeland , Timmermans Jean‐Pierre 
Published: June 2021

The intestinal wall has a complex 3D architecture, consisting of epithelial, immune, vascular, smooth muscle and neuronal components. Traditional 2D techniques are by definition destructive and therefore fail to provide a faithful 3D representation of this tissue. With an eye on visualizing intact intestinal tissue in toto, several optical clearing methods aimed at reducing optical scattering and absorption of photons, have been re‐examined or newly developed. Four hydrophilic solvent‐based clearing protocols (SeeDB2G/S, modified CUBIC, ScaleS and Ce3D) and four organic solvent‐based clearing protocols (3DISCO, iDISCO, uDISCO and Visikol) were assessed in ileal segments and their validity for in toto imaging of the intestinal wall of mice was evaluated. Optical penetration depth, conservation of endogenous GFP fluorescence, compatibility with immunolabeling and nuclear staining, and degree of preservation of the topographical architecture and cellular morphology were used as evaluation criteria. Samples were collected from CX3CR1GFP/GFP and wild‐type mice, fixed in 4% paraformaldehyde overnight and rinsed in PBS (3 × 10 min) prior to clearing. For immunostaining, the tissues were incubated up to 48 h with antibodies raised against neuronal, endothelial and/or fibroblast markers and combined with fluorescent nuclear stains such as DAPI and TO‐PRO‐3. All images were acquired using a Leica SP8/DLS confocal microscope. Visikol‐ and ScaleS‐treated intestinal samples displayed the lowest light penetration depth, while the CUBIC protocol yielded intermediate penetration depth as measured by an intensity z‐profile in the confocal microscope. The five remaining clearing protocols (iDISCO, 3DISCO, uDISCO, SeeDB2G/S, Ce3D) allowed visualization of the full‐thickness intestinal wall and did not have a significant negative impact on the antigen‐antibody binding features. However, in contrast to SeeDB2G/S and Ce3D, iDISCO, 3DISCO and uDISCO showed a number of drawbacks, including substantial tissue shrinkage and distortion of normal ENS architecture, loss of GFP signal and mucosal damage. In addition, given their highly toxic nature, these clearing procedures require a more tedious handling. The Ce3D protocol was three times faster than the SeeDB2G/S protocol. The latter two protocols appeared to give the most optimal results in visualizing the 3D architecture of the distinct components across the intestinal wall. It should, however, be kept in mind that a particular protocol successfully applied in one organ does not automatically yield a similar qualitative outcome in another organ. Therefore, optimization of clearing protocols has to be organ‐specific in terms of making the tissue transparent without having a negative impact on tissue morphology, antigenicity and immunofluorescence or preservation of GFP fluorescence.Support or Funding InformationGB was supported by a CAPES scholarship.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.