Histology and muscle fiber size determination

H&E staining was done on serial cryostat sections (6μm) of skeletal muscles (paraspinal or quadriceps femoris) according to standard procedures and examined under a light microscope. Microscopy was performed using an Olympus IX 50 inverted microscope with fluorescent attachments and images were captured with a CCD camera. 200 X images of H&E stained skeletal muscle were used for muscle fiber size determination by measuring cross-sectional area of each muscle fiber. At least five mice per group were analyzed, and for each mouse at least three different images and at least 300 fibers were analyzed. Muscle fiber size was measured using AxioVision^™ V4.8.2.0 (Carl Zeiss MicroImaging).