Ab220365

After 4 weeks of LL administration, the calf muscles were removed and fixed in 10% neutral buffered formalin. The fixed muscle tissue was embedded in paraffin, and the paraffin blocks were cut into 4 μm thick sections. To assess tissue morphology, transverse sections of muscle tissues were stained with hematoxylin (ab220365 Abcam, Cambridge, UK) and eosin (H&E). The sections were examined under a microscope (Eclipse TE 2000-U; Nikon, Düsseldorf, Germany). The PCSAs were analyzed using ImageJ software (version 2020).

Bladder tissues were embedded in paraffin wax and cut into 3 µm sections. After deparaffinization and rehydration, antigen retrieval was performed by immersing the sections in citrate buffer (pH 6.0) for 30 min in an autoclave sterilizer and then cooling at room temperature for 10 min. Sections were incubated in 3% H₂O₂ for 10 min, followed by blocking with 10% goat serum for 1 h. The blocking buffer was removed, and the sections were incubated with the primary antibody at a 1:50 dilution (Anti-ki67 antibody #ab15580, Abcam, Cambridge, England) overnight at 4 °C. Subsequently, the sections were incubated with a biotinylated goat anti-rabbit IgG secondary antibody at a 1:100 dilution, followed by incubation with diaminobenzidine solution (#ab64238, Abcam) for 1 min. The sections were counterstained with hematoxylin (#ab220365, Abcam) for 20 s, dehydrated in a series of increasing concentrations of alcohol, immersed in xylene twice for 10 min each, and coverslipped with mounting medium. The stained specimens were photographed with a TissueFAX Plus system. The Ki-67 index was determined by expressing the numbers of positively stained cells in the bladder transitional epithelium as positive cell number/mm², followed by analysis using TissueFAX Plus software.