Whole mount 3D imaged adipose tissue demonstrating sympathetic neurons and blood vessels during cold exposure and thermoneutrality

WAT, primarily comprising lipid-storing adipocytes, also contains various non-adipocyte cell types, such as neurons, immune cells or endothelial cells. Recent advancements in whole-tissue imaging suggest that the degree of sympathetic innervation and vasculature is highly heterogeneous within WAT depots. Here, we present a protocol for whole-mount WAT clearing, overcoming the constraints associated with antibody-methanol sensitivity, broadening the spectrum of applicable antibodies for spatial analysis of many different cell types. Additionally, we present TiNeQuant (Tissue Network Quantifier) a Fiji tool for automated 3D quantification of neuron- or vascular network density, freely available at https://github.com/SchweigerLab/TiNeQuant. As this easy-to-use tool is not limited to WAT, it simplifies future efforts across various fields studying neuronal or vascular alterations in numerous pathologies.