Development of an optimized clearing protocol to examine adipocyte subpopulations in white adipose tissue

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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.