Methanol h2o2

Paraffin-embedded muscle sections were deparaffinized using xylene (Junsei, Tokyo, Japan), rehydrated using an ethanol gradient, and treated with methanol/H₂O₂ (Junsei) to quench endogenous peroxidase activity. Muscle sections were blocked with 1% normal goat serum (SeraCare Life Sciences, Milford, MA, USA) and incubated with protein-specific antibody [FMOD, MSTN, MYOG, MuRF1, Atrogin1, RAGE, Glb1, PPARγ, CD36, and CD163 (1:50)] overnight at 4 °C. Sections were then treated with HRP-conjugated secondary antibody (1:100; Santa Cruz Biotechnology), incubated for 1 h at room temperature, counterstained with hematoxylin, dehydrated, mounted, and examined under an optical microscope (Leica, Seoul, Korea). Morphological changes were examined in hematoxylin and eosin-stained sections under an optical microscope (Leica, Wetzlar, Germany).

Muscle tissues were routinely fixed with 10% formalin and embedded in paraffin wax [60 (link)]. Paraffin-embedded tissue sections were deparaffinized, hydrated, and endogenous peroxidase was quenched using xylene (Junsei, Tokyo, Japan), an ethanol gradient, and methanol/H₂O₂ (Junsei), respectively. After blocking with 1% normal goat serum, sections were incubated with primary antibody [Pax7 (1:50), MYOD (1:50), MYOG (1:50), MYL2 (1:50), MSTN (1:50), MuRF1 (1:50), Atrogin1(1:50), nitrotyrosine (1:50)] overnight at 4 °C, and then treated with HRP-conjugated secondary antibody (1:100; Santa Cruz Biotechnology) at room temperature for 1 h. Sections were then counterstained with hematoxylin, dehydrated, mounted, and examined under a light microscope (Leica, Seoul, Korea). After Hematoxylin and Eosin staining (H&E, Thermo Fisher Scientific), muscle tissues were examined for morphological changes under a light microscope.