Frozen Skin Tissue Block Preparation: A Protocol to Preserve Cutaneous Melanoma Samples from Murine Dorsal Skin

- Quiescent melanocytes stem cells, in response to UV radiation or hair depilation, get activated. Activated melanoma stem cells can lead to cutaneous melanoma - a form of skin cancer. To study melanoma, we prepare frozen skin tissue blocks to retain the native structures of DNA and proteins during the fixing procedure.

Begin by placing a euthanized melanoma mouse on a surgical bed and remove hair from the dorsal region of the skin above the tail. Excise the skin and cover it in a filter paper. Next, submerge the filter paper containing skin tissue in neutral buffered formalin for the desired time. Formalin penetrates the skin tissue and binds to the amino acids, which causes the cross-linking of proteins, therefore leading to tissue fixation.

Remove the skin tissue from paper and cut the tissue to obtain pieces of the desired size. Now, embed the tissue pieces vertically in a cryomold filled with OCT medium and place the cryomolds on dry ice. OCT is a specimen matrix that solidifies on freezing and forms a gelatinous layer around the tissue specimen holding it in place. Use the frozen blocks for further histological assays. In the following protocol, we will prepare frozen skin tissue blocks from the murine dorsal skin to study cutaneous melanoma.

- To isolate the skin samples, use an electric trimmer to remove any hair from the dorsal skin area of the euthanized irradiated mice and lightly brush the exposed skin with a lint-free tissue to clear the area. Make a small incision with sharp scissors just above the base of the tail and insert large blunt scissors into the incision to separate the subdermal connective tissues. Using sharp scissors, carefully cut along the edge of the skin region of interest to isolate the tissue sample, and place the excised skin tissue onto a clean paper towel.

Then, use dull forceps to stretch the skin so that it adheres to the towel and is taut. When all of the samples have been harvested, place the skin sample and paper towel backing into a piece of folded filter paper immediately adjacent to the crease, taking care that the sample is smooth and not folded or crumpled. Close the sides of the filter paper with staples and trim the paper. Then, fully submerge the samples in 10% neutral buffered formalin for 3 to 5 hours at room temperature before washing the tissues with two 5 minute washes in deionized water.

After the second wash, carefully remove any residual material from the skin tissues and trim the edges of the sample so that they are not jagged. Next, make three sagittal cuts in each sample so that the width of the strips is no more than 5 millimeters and make transverse cuts so that the samples are no more than 20 millimeters wide. Position a cryomold containing optimum cutting temperature or OCT medium in a portrait orientation and place four to five pieces of skin on top of the OCT.

When all of the pieces are in place, use two pairs of fine forceps to bring the long edge of each piece of skin to the bottom of the cryomold so that the samples are vertical within the OCT and perpendicular to the base of the mold. Then, fill the mold with additional OCT to the second lip, and place the mold onto a flat surface of a piece of dry ice, using forceps to adjust any skin pieces that may have shifted during the transfer as the block begins to freeze as necessary.