2.10. Microscopic evaluation of H&E-stained samples
On days 3, 7, 14, and 21 after surgery, the rats were sacrificed, and the wound samples together with a surrounding rim of normal skin were harvested. For histological analysis, the samples were first fixed in 4% neutral buffered paraformaldehyde, sequentially dehydrated in a series of ethanol with increasing gradient concentration, and cleared by the exposure to xylene. Then, the samples were incubated with paraffin xylene solution in an oven overnight, to remove the xylene, and embedded in paraffin. The paraffin-embedded samples were sectioned at the thickness of 8.0 μm to adhere to glass slides. After the process of dewaxing and hydration, the sections were stained with H&E for routine examination. Then, Masson′s trichrome staining was performed to further analyze the characteristics of these wound samples. Sections were analyzed, and images were captured by microscopy (Olympus IX71, Tokyo, Japan). H&E stained samples were used to measure the thick ratio of epithelial thickness at 14 and 21 d post-wounding.