Summary Little is known about the diversity and function of adipose tissue nerves, due in part to the inability to effectively visualize the tissue’s diverse nerve subtypes and the patterns of innervation across an intact depot. The tools to image and quantify adipose tissue innervation are currently limited. Here, we present a method of tissue processing that decreases tissue thickness in the z-axis while leaving cells intact for subsequent immunostaining. This was combined with autofluorescence quenching techniques to permit intact whole tissues to be mounted on slides and imaged by confocal microscopy, with a complementary means to perform whole tissue neurite density quantification after capture of tiled z-stack images. Additionally, we demonstrate how to visualize nerve terminals (the neuro-adipose nexus) in intact blocks of adipose tissue without z-depth reduction. We have included examples of data demonstrating nerve subtypes, neurovascular interactions, label-free imaging of collagen, and nerve bundle digital cross-sections.

53Adipose tissue requires neural innervation in order to regulate important metabolic functions. 54Though seminal work on adipose denervation has underscored the importance of adipose-nerve 55 interactions in both white (energy storing) and brown (energy expending) adipose tissues, much 56 remains a mystery. This is due, in part, to the inability to effectively visualize the various nerve 57 subtypes residing within these tissues and to gain a comprehensive quantitation of neurite 58 density in an entire depot. With the recent surge of advanced imaging techniques such as light 59 sheet microscopy and optical clearing procedures, adipose tissue imaging has been 60 reinvigorated with a focus on three-dimensional analysis of tissue innervation. However, 61 clearing techniques are time consuming, often require solvents caustic to objective lenses, alter 62 tissue morphology, and greatly reduce fluorophore lifespan. Not only are current methods of 63 imaging wholemount adipose tissues inconvenient, but often attempts to quantify neurite density 64 across physiological or pathophysiological conditions have been limited to representative 65 section sampling. We have developed a new method of adipose tissue neurite imaging and 66 quantitation that is faster than current clearing-based methods, does not require caustic 67 chemicals, and leaves the tissue fully intact. Maintenance of a fully intact depot allowed for tiling 68 z-stacks and producing maximum intensity projections of the entire adipose depot, which were 69 then used to quantify neurite density across the tissue. With this processing method we were 70 able to characterize the nerves, nerve-subtypes, and neurovascular interactions within the 71 inguinal subcutaneous white adipose tissue in mice using up to five fluorescent channels at high 72 resolution. We also utilized second harmonic generation, which provides label-free imaging, to 73 investigate collagen fiber abundance in adipose of obese mice. 74 75 76 77 78 79 80 81 82 83 84 85 86 87 Introduction: 88Historically overlooked as a location of diverse peripheral innervation [1], the adipose organ 89 was most prominently inspected for innervation in the mid 1960's when sympathetic nerve fibers 90 were visualized within brown adipose tissue (BAT) [2]. BAT nerves were later comprehensively 91 investigated by T.J. Bartness [3]. Energy expending BAT was the first adipose tissue to be 92 identified as being highly innervated due to its important role in thermogenesis [4], which 93 requires significant sympathetic input [2, 5, 6]. Sensory innervation has also been documented 94 in BAT, particularly around vasculature, and has been proposed to play a role in lipolysis [7]. 95More recently white adipose tissue (WAT), associated more with energy storage, was 96 demonstrated to be highly innervated by sympathetic [8, 9] and sensory nerves [5, 10], but not 97 parasympathetic [11] nerves. 98In order to visualize adipose innervation, it had been common practice to slice adipose 99 tissue into 7um-10um thick sections and...