Abstract
Organic solvent dibenzyl ether (DBE)-based protocols have been widely used in adipose tissue clearing. However, benzyl alcohol/benzyl benzoate (BABB)-based clearing has been shown to offer better transparency in other tissues. The addition of diphenyl ether (DPE) to BABB (BABB-D4) is often included to preserve fluorescent signals, but its effects on adipose tissue transparency and shrinkage have not been explored. Distinct adipocyte subpopulations contribute to its cellular composition and biological activity. Here, we compared clearing solvents to create an optimized clearing methodology for the study of adipocyte subpopulations. Adipose tissues were cleared with BABB, BABB-D4, and DBE, and post-clearing transparency and tissue shrinkage were measured. An optimized protocol, including BABB-D4 clearing, delipidation, and extensive immunofluorescence blocking steps, was created to examine the spatial distribution of Wt-1 positive progenitor-derived (Type-1) adipocytes in intact mesenteric fat. Both BABB and BABB-D4 lead to significantly increased tissue transparency with reduced tissue shrinkage compared to DBE-cleared adipose tissue. Type-1 adipocytes are found in a clustered distribution with predominant residence in fat associated with the ileum and colon. This paper details an optimized clearing methodology for adipose tissue with increased tissue transparency and reduced shrinkage, and therefore will be a useful tool for investigating adipose tissue biology.
Highlights
Traditional immunohistochemistry approaches rely on mechanical sectioning and analyses of small sections, which limit the interpretation of 3D cellular microenvironments
Focus stacking of images taken by confocal microscopy has been widely used to study adipose structures in physiological and pathological conditions; the depth of visualization is usually only a few hundred micrometers due to refractive index mismatch [1]
We found that Type-1 adipocytes were highly concentrated in fat regions associated with the ileum and colon, but to a lesser extent in the duodenum and jejunum, and that Type-1 adipocytes exhibited a clustered distribution within these regions of mesenteric fat
Summary
Traditional immunohistochemistry approaches rely on mechanical sectioning and analyses of small sections, which limit the interpretation of 3D cellular microenvironments. Advances in imaging technology such as confocal and multiphoton microscopy replace mechanical sectioning with optical sectioning. Optical clearing methods have been developed to match refractive indices of tissues to the surrounding media and enable deep-tissue imaging. Three clearing method types (aqueous-, hydrogel-, or solvent-based) each come with advantages and disadvantages. Aqueous-based clearing methods provide biocompatibility, biosafety, and high degree of fluorescence preservation [2,3]. They have reduced optical clearing power compared to other techniques [4]. Solvent-based methods are fast and provide high degree of tissue transparency, but quickly quench fluorescence and require utilization of toxic chemicals [1]
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