Concept

In traditional approaches to tissue clearing, a variety of methods are used to remove lipids from the sample. However, the majority of the proteins, DNA, RNA and other biomolecules remain in place. As each of these components has a different refractive index, this leads to scattering and restricts the level of clarity that standard tissue clearing methods can achieve.

In replication of endogenous patterns via label incorporation in chemoablated acrylamide (REPLICA), we remove all of the sample. Antibodies, or other fluorescently labelled binding agents, are functionalised with vinyl groups, such that they can participate in the free radical polymerisation of a polyacrylamide hydrogel. After staining and polymerisation, the binding agents are now locked in their 3D positions by covalent incoporation into the hydrogel network and so form a fluorescent footprint of the original target locations even after the targets themselves have been removed in the sample stripping steps. Hence, after completing the protocol, we now have a fluorescent replica of the original sample.