Pad418mu02

Muscle fiber type and Pax7-positive nuclei were determined using the ImmunoCruz^® rabbit ABC Staining System (sc-2018; Santa Cruz Biotechnology, Dallas, TX, USA) according to the manufacturer’s guidelines. Briefly, 8 μm frozen muscle sections were preincubated for 5 min in H₂O₂ and washed twice for 5 min in PBS and blocked for 1 h at room temperature. Subsequently, the sections were incubated overnight at 4 °C with primary antibodies for type 1 muscle fiber (1:500; PAD418Mu02; Cloud-Clone Corp, Katy, TX, USA), type 2A muscle fiber (1:500; PAA755Mu01; Cloud-Clone Corp), type 2B muscle fiber (1:500; PAD416Mu01; Cloud-Clone Corp), and Pax7 (1:100; AP10488B; Abcepta, San Diego, CA, USA). On the next day, sections were washed three times for 5 min in PBS and incubated with biotinylated secondary antibody for 1.5 h. After washing in PBS, the sections were incubated for 30 min with AB enzyme reagent. Sections were then washed and incubated in three drops of peroxidase substrate for 5 min. Finally, sections were washed in deionized H₂O for 5 min, dipped in 90/95/99.5% ethanol and xylene, and mounted with coverslips using mounting reagent (NEW M·X, Tokyo Garasu Kikai, Tokyo, Japan). Tissue slides were observed using PALM MicroBeam IV (ZEISS). The fiber-type distribution and number of Pax7-positive nuclei per muscle fiber was calculated from 200 fibers using the ImageJ software.