Fig. 1

Workflow for tissue clearing and staining. a Before the clearing process, tissue is prepared for clearing by cross-linking proteins and nucleic acids using epoxides. This cross-linking provides increased structural stability in preparation of lipid removal from the tissue [62]. Lipids are washed from the tissue using sodium dodecyl sulphate (SDS). The fully cleared tissue can be fluorescently labeled and incubated in an index of refraction matching solution so that its refractive index matches that of a glass cover-slip. b Passive tissue clearing is diffusion based using a floating sections protocol. c Fluorescent molecules diffuse to their target sites in the floating section. d After the fluorescent dyes have been fixed in place and the sample has been refractive-index (RI) matched to the glass coverslip, the tissue becomes translucent. Fluorophores can be visualized at any point in the Z-dimension of the respective samples. Created with www.BioRender.com