Fitc f0257

Vastus lateralis samples of 148 physically active participants were obtained from the left leg using the modified Bergström needle procedure with aspiration under local anaesthesia with 2% lidocaine solution. Prior to analysis, samples were frozen in liquid nitrogen and stored at − 80 °C. Serial cross-sections (7 μm) were obtained from frozen samples using an ultratom (Leica Microsystems, Germany). Sections were thaw-mounted on Polysine glass slides, maintained at room temperature (RT) for 15 min and incubated in PBS (3 × 5 min). The sections were then incubated at RT in primary antibodies against slow or fast isoforms of the myosin heavy chains (M8421, 1:5000; M4276; 1:600, respectively; Sigma-Aldrich, USA) for 1 h and incubated in PBS (3 × 5 min). Next, the sections were incubated at RT in secondary antibodies conjugated with FITC (F0257; 1:100; Sigma-Aldrich) for 1 h. The antibodies were removed, and the sections washed in PBS (3 × 5 min), placed in mounting media and covered with a cover slip. Images were captured by fluorescent microscope (Eclipse Ti-U, Nikon, Japan). All analyzed images contained 329 ± 14 fibers. The ratio of the number of stained fibers to the total fiber number was calculated. Fibers stained in serial sections with antibodies against slow and fast isoforms were considered hybrid fibers.