Fluorescent Staining and Clearing Protocol for Liver Cuboids

Liver cuboids embedded in collagen were stored in 30% sucrose solution prior to staining and clearing. Cuboids were first dehydrated in graded steps to a 70% ethanol solution (v/v in water). Samples were then stained with a fluorescent H&E analog consisting of a 1:500 dilution of TO-PRO3 Iodide (Cat: T3605, Thermo-Fisher) and 1:4000 dilution of Eosin-Y (Cat: 3801615, Leica Biosystems).^{55 (link)} Following staining, samples were fully dehydrated in 100% ethanol and then cleared and stored in ethyl cinnamate at room temperature before and after imaging (Cat: 112372, Sigma-Aldrich).^{57 (link)}