For histological evaluation, TA muscle samples harvested from mice after 4 weeks were fixed with 4% paraformaldehyde at 25 °C for 24 h. The samples were then paraffin-embedded and sectioned into 5 μm sections. Subsequently, deparaffinized sections were stained with hematoxylin and eosin (H&E) and MT for histological analysis. The diameters of the muscle fibers and collagen areas were quantified using ImageJ software.
Additionally, the deparaffinized sections were treated with mouse anti-MHC (1:50 dilution; Santa Cruz Biotechnology, Dallas, TX, USA) and rabbit anti-human leukocyte antigen (HLA; species reactivity: human) (1:50 dilution; Abcam, Cambridge, UK). The muscle sections were then rinsed with PBS three times, immersed in Alexa Fluor 488-conjugated anti-rabbit (1:200 dilution; Abcam) and Alexa Fluor 594-conjugated anti-mouse (1:200 dilution; Abcam) secondary antibodies for 30 min, and counterstained with DAPI. Stained sections were visualized using a confocal microscope. The percentage of HLA + myofibers was quantified using ImageJ software in a blinded manner.
Additionally, the deparaffinized sections were treated with mouse anti-MHC (1:50 dilution; Santa Cruz Biotechnology, Dallas, TX, USA) and rabbit anti-human leukocyte antigen (HLA; species reactivity: human) (1:50 dilution; Abcam, Cambridge, UK). The muscle sections were then rinsed with PBS three times, immersed in Alexa Fluor 488-conjugated anti-rabbit (1:200 dilution; Abcam) and Alexa Fluor 594-conjugated anti-mouse (1:200 dilution; Abcam) secondary antibodies for 30 min, and counterstained with DAPI. Stained sections were visualized using a confocal microscope. The percentage of HLA + myofibers was quantified using ImageJ software in a blinded manner.