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 confocal microscope to visualize fluorescently-labeled regions of interest in the three-dimensional sample. The following protocol allows the visualization of keratinocyte proliferation markers' expression in whole mount skin biopsies using the CUBIC protocol.

- Begin by using trimmers to shave the hair from the neck of a euthanized mouse, taking care not to wound the skin. Then, decontaminate the skin with 70% ethanol in PBS. Next, lift the dorsal neck skin with forceps and use scissors to remove an approximately 1.5 by 4 centimeter area of dorsal mouse skin. Then, flatten the skin, dermis side down, on a piece of filter paper, making note of the anterior-posterior orientation of the sample and trim the paper around the dissected skin.

- For optimal imaging, the skin samples need to remain flat with consistent hair follicle orientation. To maintain samples in a proper antero-posterior position, we fix the skin pieces onto filter paper in rectangular biopsies.

- Transfer the samples to a 15 milliliter tube filled with freshly prepared 4% PFA in PBS. Then, when they have been firmly fixed to the filter paper, transfer the specimens to a new 15 milliliter tube of PBS for two 5-minute washes.

To clear the skin biopsies after the second wash, use a sharp razor blade to cut the tissues into approximately 0.2 by 0.5 centimeter pieces, taking care that the longer sides of the samples are cut along the anterior-posterior direction of the samples.

- Cutting the longer side of the biopsy parallel to the orientation of the hair follicles also helps avoid extensive hair follicle damage.

- Then, submerge the biopsies in 5 milliliters of freshly prepared CUBIC1 clearing solution in a new 15 milliliter tube and place the tube on a rotating platform in a hybridization oven at 37 degrees Celsius. Once the tissue pieces are transparent, wash the biopsies in 4 milliliters of PBS for four 6-hour washes at 37 degrees Celsius followed by a 4-hour 37 degrees Celsius wash in 20% weight per volume sucrose in PBS.

At the end of the incubation, freeze each sample in optimal cutting temperature compound in individual 15 milliliter tubes overnight at minus 80 degrees Celsius to increase the permeability of the tissues to antibody penetration.

The next morning, stain the skin samples with the appropriate antibodies and vital dyes of interest. Then, incubate the specimens in 1 milliliter of freshly prepared CUBIC2 clearing solution in 2 milliliter tubes on a shaker for 24 hours in the 37 degrees Celsius oven to even the refractive index of the tissues.

When the tissues are clear, position the biopsies along the longer side of individual glass cover slips such that the direction of the hair follicle growth is parallel to the cover slip surface. Put one drop of CUBIC2 solution over the tissue. Place two 1 millimeter by 2 centimeter strips of blue tack on a cover slip. Then, cover the biopsy with a second cover slip.

Next, place the imaging chamber onto a confocal microscope stage and move the tissue into the light pathway. Using the appropriate light source and standard epifluorescence filters, scan the sample to identify fluorescently stained regions of interest. Then, acquire fluorescent confocal images of the regions of interest.