A17069

The sections cut from soleus muscle tissue were deparaffinized and subjected to antigen retrieval under high temperature and pressure. The sections were then blocked with 5% skim milk powder for two hours and subsequently incubated with primary antibodies against IL-1β (1:250, SC-12742, Santa Cruz), TNF-α (1:250, SC-52746, Santa Cruz), Drp1 (1:200, A17069, ABclonal), and Fis1 (1:200, A19666, ABclonal) overnight at 4 °C. After rinsing off the primary antibodies, the sections were incubated with the corresponding secondary antibodies for 2 h. The target proteins in the soleus muscle sections were then visualized using fluorescently labeled tyramine (G1222, Servicebio, China), washed with TBST, and counterstained with DAPI (G1012, Servicebio, China). Images were acquired with a confocal microscope (Carl Zeiss AG, Germany) and analyzed quantitatively using Image-J software.

Protein concentrations in 30 mg of soleus muscle tissue extracted from the right limb of rats were measured using a BCA kit. Subsequently, 45 µg of protein, dissolved in loading buffer, was subjected to electrophoresis and then transferred onto polyvinylidene difluoride (PVDF) membranes. After blocking with 5% skim milk powder, the PVDF membranes were incubated with primary antibodies against IL-1β (1:1000, SC-12742, Santa Cruz), TNF-α (1:1000, SC-52746, Santa Cruz), Drp1 (1:2000, A17069, ABclonal), Fis1 (1:2000, A19666, ABclonal), MFF (1:2000, GB114102, Servicebio), MFN2 (1:5000, 12186-1-AP, Proteintech), FNDC5 (1:2000, A18107, ABclonal), AMPK (1:500, A12718, ABclonal), and p-AMPK (1:1000, AP0432, ABclonal) for 12 h at 4 °C. This was followed by incubation with the appropriate secondary antibodies. The expression of the target proteins was analyzed using ImageJ software, with β-actin (1:50,000, AC048, ABclonal) serving as a reference.