Comparison of Clearing Methods to Visualize Diabetic Neuropathy in BTBR Ob/Ob Mice

Clearing methods are used to render a tissue completely transparent by removing the light scattering lipids. Many of these methods have been optimized for mouse brains, the fattiest organ in the body next to adipose tissue. In this study we aimed to test and optimize multiple tissue clearing methods and microscopy techniques on adipose tissue samples, to be able to visualize the neural innervation using either endogenous reporters (ie: tyrosine hydroxylase, which marks activation of sympathetic nerves) or immunostaining for nerves and synapses. The novel ability to visualize the entire neural arborization in the adipose depots under conditions that we have shown to induce adipose neural plasticity (cold exposure, exercise, etc) or promote adipose neuropathy (death of nerves, such as with diabetes, aging, obesity) will provide insight into nerve dynamics that would not be possible through traditional methods like 7-10 micron tissue cross sections. We started with the CLARITY clearing method that was originally optimized for brain, then Scale View A2, CUBIC and CUBIC CB, BABB clearing, and have yet to test iDisco and its alternative- uDisco. Of the clearing methods tested, CUBIC CB provided fast and effective clearing that worked well for immunostaining and will now be further tested on endogenous reporters.

Faculty Mentor: Kristy Townsend

Co-authors: Amanda Dubois, Magdalena Blaszkiewicz