CUBIC Protocol Visualizes Protein Expression at Single Cell Resolution in Whole Mount Skin Preparations

The overall goal of this procedure is to visualize cell proliferation and protein expression patterns at the single cell level in three dimensions and to accurately assess the anatomy and pathology of the skin. This method can help answer key questions about how cellular and molecular mechanisms control epidermal development and regeneration and how these mechanisms are perturbed during skin disease. The main advantage of this technique is that it is a technically simple protocol for visualizing individual cells in full thickness skin biopsies in three dimensions with an unprecedented accuracy.

The method also facilitates the investigation of the interactions between the epidermis and the dermis in full skin samples. Generally researchers new to this method might struggle a bit in preparing the small flat skin biopsies without damaging the hair follicles. Demonstrating the procedure will be Betty Maclarios, a Ph.D.student at the Developmental and Regenerative Dermatology Unit at UNSW Australia.

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 and PBS. Next lift the dorsal neck skin with forceps and use scissors to remove an approximately 1.5 by 4 cm 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 the proper anterior posterior position we fix the skin pieces on the filter paper in rectangular biopsies.

Transfer the samples to a 15 mL tube filled with freshly prepared 4%PFA and PBS. Then when they have been firmly fixed to the filter paper transfer the specimens to a new 15 mL tube of PBS for two five 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 cm pieces taking care that the longer sides of the samples are cut along the anterior posterior direction of the samples.

Cutting along the side of the biopsy parallel to the orientation of the hair follicles also helps avoid extensive hair follicle damage. Them submerge the biopsies in 5 mL of freshly prepared cubic one clearing solution in a new 15 mL 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 mL of PBS for four six hour washes at 37 degrees Celsius followed by a four hour 37 degree Celsius wash in 20%weight per volume sucrose and PBS.

At the end of the incubation freeze each sample in optimal cutting temperature compound in individual 15 mL tubes overnight at 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 mL of freshly prepared cubic two clearing solution in 2 mL tubes on a shaker for 24 hours in the 37 degree 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 cubic two solution over the tissue. Place two 1 mL by 2 cm 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. Using this method both thickness dorsal skin biopsies of adult wild type mice can be clarified and stained with antibody against the basal keratinocyte marker keratin 14.

Dappy positive nuclei are visible throughout the sample and allow appreciation of some of the anatomical features such as the dermal papillae. The K14 staining is apparent in the one cell thick basal layer of the interfollicular epidermis. Outlining the sebaceous glands and the outer root sheaths of the hair follicles and in the secondary hair germs with only low levels of K14 expression detected in the bulge area of the hair follicles.

To visualize the proliferating cells full thickness dorsal skin biopsies at telogen of adult wild type mice can be clarified and stained as demonstrated revealing the presence of proliferating keratinocytes in the basal interfollicular epidermis and in the isthmus but not the bulge region of the hair follicles. To visualize the sebaceous glands full thickness dorsal skin biopsies at antigen of adult wild type mice can be clarified and stained, facilitating the detection of the sebaceous glands in the isthmus region of the hair follicles. To visualize morphological changes in the epidermis and transgenic animals full thickness dorsal skin biopsies can be clarified and stained revealing hyperplasia of the interfollicular epidermis and abnormal hair follicles with K14 labeled cell masses extending proximally into the dermis representing the enlarged transgenic stem cell populations.

After watching this video we should have a good understanding of how to prepare and clarify skin samples and to visualize protein expression patterns at the single cell resolution in three dimensions. This method is simple to perform and uses relatively safe and inexpensive reagents. In the future more antibodies will be tested for their compatibility with this method allowing this analysis to be extended to visualize more proteins and certain types of interest.

Other imaging methods such as light sheet microscopy can be performed for visualization of skin anatomy and protein expression patterns of larger skin samples in three dimensions. After its development this technique will pave the way for researchers in the field of dermatology to explore the cellular and molecular interactions between the dermis and epidermis in skin homeostasis, in genetically modified mouse models and in one of our models of skin disease.