Caseviewer software version 2.3

HCT116 and HCT15 cells were cultured on glass coverslips attached to a six-well plate. Then, 4% paraformaldehyde was used to fix the cells, and 0.3% Triton X-100 was used to enhance the permeability of the cell membranes. Then, the cells were incubated in 5% bovine serum albumin for 1 h and incubated in the corresponding primary antibody at 4 °C overnight, followed by incubation with the secondary antibody at room temperature for another 2 h. DAPI (300 nM) staining was employed for nuclear localization. The fluorescence staining of the cells was visualized using a fluorescence microscope (Nikon, Japan). The fluorescent images were acquired using CaseViewer software (version 2.3) (3DHISTECH, Budapest, Hungary) and analyzed with ImageJ software (version 1.8.0).

The tissue sections were stained with hematoxylin and eosin, and images were collected using a Panoramic SCAN (3DHISTECH Ltd., Hungary) at the Kaohsiung Chang Gung Memorial Hospital. For different tattoos, variations in the average epidermal and dermal thickness were measured using CaseViewer software version 2.3 (3DHISTECH Ltd. Hungary).
For the histological study of repaired tissues at 2 and 6 months post-wound creation, a scalpel was used to excise a long rectangular piece of tissue from the center of each wound or normal skin. The samples were immersed and fixed in 10% formalin buffer for 24 h, dehydrated, and embedded in paraffin. Samples were sectioned at a 5-µm thickness and stained with hematoxylin and eosin (Merck, Germany) to observe the skin structure; picrosirius red stain (Polysciences Inc., USA) was used to identify the types and distribution of collagen fibrils, Masson’s trichrome stain HT-15 (Sigma-Aldrich Co., Switzerland) was used to determine the architecture and biosynthesis of collagen, and Verhoeff’s stain (Polysciences Inc., USA) was used to clarify the distribution and maturation of elastin fibrils. The stained tissue sections were analyzed under a light microscope (Leica, Germany) or with a polarizer when picrosirius red images were observed.