Until recently, adipose tissue was conceived of as an amorphous collection of fat cells. Over the past few decades, our understanding has grown more sophisticated, with fat now recognized to be a complex organ containing different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, the vasculature, and nerve projections. Interactions among these adipose-resident cells have pronounced effects on adipose tissue and organismal physiology and pathophysiology1. Although emerging studies have unraveled important molecular mechanisms underlying certain interactions, a more comprehensive understanding requires reliable structural profiling of the entire tissue in three dimensions (3D).
Our current knowledge of adipose tissue morphology is largely based on histological analysis of thin sections (5 μm) with relatively high-magnification imaging (more than 10X)2,3. However, this approach has several significant limitations. First, intricate filamentous structures such as sympathetic nerves and the vasculature, which are known to play important roles in adipose function4,5,6,7, are difficult to evaluate through thin sections. Second, due to its seemingly amorphous shape and the lack of representative structural units to focus on, it is difficult to appreciate adipose tissue structures based only on section staining. Third, adipose tissue has a very high lipid content, creating challenges in obtaining consistent serial sections that are suitable for 3D anatomical reconstruction, a conventional method used to study whole brain morphology8. Given these factors, there is a great need for a whole-mount approach that can provide 3D visualization of an entire adipose depot while still achieving cellular resolution.
3D volumetric imaging of an entire organ is challenging due to the obscuring effects of light scatter. A major source of light scatter in biological tissues comes from lipid-aqueous interfaces. Although the efforts to eliminate scatter by removing lipids have been ongoing for over a century, there have been a large number of recent innovations9. One such newly developed tissue-clearing method is immunolabeling-enabled 3D imaging of solvent-cleared organs (iDISCO/iDISCO+)10,11. However, adipose tissue presents a particular challenge given its high level of lipids, and therefore, additional modifications to the iDISCO/iDISCO+ protocol are required to fully extract the lipids while protecting the tissue from collapsing. The modified protocol we have developed, now called Adipo-Clear, employs methanol/dichloromethane-based delipidation of adipose tissue to achieve optimal transparency suitable for high-resolution volumetric imaging12. Because the delipidation step largely quenches endogenously expressed fluorescent proteins such as GFP and RFP, visualization of such proteins must be achieved by immunolabeling. Overall, this simple and robust protocol can be applied to study tissue-level organization of adipose-resident cells, lineage tracing of adipocyte progenitor cells, and adipose morphogenesis during development.
This method can help answer key questions in the field of Adipose Tissue Biology, such as how different components of Adipose Tissue interact in an entire tissue. The main advantage of this technique is that it fully removes the excess lipids stored in Adipose Tissue while maintaining it's three-dimensional architecture. While this method can provide insight into those three-dimensional organization of Adipose Tissue it can also be generally applied to other tissues with high lipid content such as memory gland and lymph node.
Visual demonstration of this method is critical as the delipidation and the clearing steps require sufficient incubation times, which can be difficult to determine. Begin by using blunt ended forceps to dissect the fat pad of interest from a freshly perfused animal model. Carefully remove the entire fad pad without pinching or squeezing the tissue or contamination the dissected tissue with fur.
Place the fat pad in approximately 10 milliliters of fixation solution in a 15 milliliter conical tube and post fix at four degrees celsius overnight. The next day after washing the sample with PBS, gently remove any debris from the sample under a dissection microscope. H