CUBIC-based Protein Expression Visualization: A Procedure for Visualizing Protein Expression in Full Thickness Mouse Skin Biopsies

- Skin is a complex organ consisting of varied cell populations arranged in multiple layers. CUBIC, a tissue clearing-based three-dimensional imaging technique, helps visualize protein expression patterns inside these skin cells using whole skin biopsies. Begin by taking a euthanized mouse and removing hair from its dorsal neck region. Excise a piece of skin and cut it into small pieces.

Submerge the pieces into a CUBIC1 clearing solution and incubate. Skin cells are non-transparent, owing to the presence of chromophores and lipids that cause light scattering. The chemicals in the CUBIC solution remove these molecules, thus reducing light scattering and making the tissue transparent.

Next, treat the tissue with the desired primary antibodies that bind specifically to their target intracellular proteins expressed in skin cells. Further, treat with fluorescently-labeled secondary antibodies that bind to the protein-primary antibody complexes. Counterstain the tissue with a DNA-binding dye that stains the nucleus.

Finally, immerse the sample in a CUBIC2 solution. The solution further reduces light scattering within the tissue, making it more transparent for imaging. Observe under a co