4.6. Histological Analysis

Gastrocnemius muscles were fixed in 10% buffered formalin for 24 h at 4 °C, dehydrated through graded alcohols, and embedded in paraffin. The muscles were sectioned into 5 μm slices using a microtome (Leica RM2235, Wetzlar, Germany). Hematoxylin-eosin (H & E) and Sirius red (G1470, Solarbio, Beijing, China) staining were performed to examine histological alteration and collagen deposition, respectively. The procedure was performed according to the manufacturer’s protocol. Before staining, paraffin sections were deparaffinized using xylene and hydrated with distilled water. For H & E staining, section-mounted glass slides were stained in hematoxylin for 5 min and then washed in distilled water. Slides were further immersed in an eosin solution for 1 min and washed in distilled water for 10 min. After 1 min, the slides were sequentially immersed in a series of ethanol solutions (70%, 95%, and 100%), for dehydration. After immersion in xylene for 3 min twice, the slides were fixed with neutral resin and sealed with cover slip. For Sirius red staining, we used the Mayer hematoxylin for 10 min, followed by washing. Then the collagen was stained by Sirius red for 1 h. The other steps were the same as the H & E staining’s processes. Images were captured with an Olympus microscope system (IX73P2F, Olympus, Tokyo, Japan).