Histological Analysis of Skin Wound Healing

After the experiments, the experimented mouse or pig skin was cut off. The targeted area was the incised skin. Skin tissues were embedded in the Tissue‐Tek optimal cutting temperature compound and cryosectioned into 5‐µm slices. The tissue slabs were processed by standard histological procedures, histochemically stained with hematoxylin and eosin (H&E), F4/80 antibody, trichrome, and CD³⁺ antibody.^[105, ^106] Antibodies were diluted according to manufacturer's instruction, unless indicated otherwise. Serving as a control, two wounds were created on the dorsal side of each mouse by removing full‐thickness skin via 3‐mm punch biopsy. Tegaderm films (3 M Inc.) were used to cover the wounds and prevent water loss in all the mice until the wounds were fully epithelialized. At days 7 and 14 postsurgery, three mice in each group were euthanized and the wounded skin removed, fixed in formalin, embedded in paraffin, and sectioned. Trichrome, F4/80 antibody, and CD³⁺ antibody staining were used for histological observations. Microscopic evaluation of the tissue sections was performed after that. The sections with F4/80, and CD³⁺ staining were observed under the microscope (Eclipse Ti2; Nikon Inc.) at 20× and 200× magnification. Four fields were randomly selected from each section to count the F4/80 positive macrophages, and CD³⁺ positive T‐cells. Immunoreactive cells were quantified as the mean cell count expressing the appropriate positive marker per high‐power field (HPF). Histological data were expressed as mean ± standard deviation (SD). Statistical analysis was performed by Student's t‐test. A p value of < 0.05 was considered significant.