Immunostaining of Muscle Tissue Sections

Briefly, frozen sections of TA muscles with a thickness of 5 μm were fixed in cold acetone for 5 min, followed by air-drying for 30 min. The tissues were then rinsed twice with Tris-buffered saline containing 0.05% tween-20 (TBS-T). Subsequently, the sections were treated with Dako Protein Block (serum-free, Cat#X0909) for 30 min at room temperature to block any nonspecific binding. After the blocking step, the sections were incubated overnight at 4 °C with primary antibodies diluted in Dako Antibody Diluent (Cat# S3022). The sections were then washed three times for 5 min each with TBS-T, followed by a 30-min incubation at room temperature with Alexa Fluor-conjugated secondary antibodies (either 488 anti-mouse or 546 anti-rabbit, both used at a 1:600 dilution, Invitrogen). After another round of washing with TBS-T three times, the sections were stained with DAPI (Thermo Fisher Scientific, D1306) in TBS-T for 5 min. Fluorescence images were captured using the NIS-Elements system (Nikon)^{46 (link)}. The antibodies used for both western blotting and immunostaining are listed in the Key Resources Table. To evaluate differences in the cross-sectional areas (CSA) of myofibers, 5 μm sections of TA muscles were stained with an anti-laminin antibody. The CSA of 300 myofibers per TA muscle was measured using Image J software (National Institutes of Health).