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For muscle analysis, mice we sacrificed 24 h later and muscles were dissected, fixed in paraformaldehyde (PFA) and mounted on slides.
While some scAAV9-treated dKO mice were kept for long-term analysis, six were sacrificed at 12 weeks of age (9 weeks after injection) for muscle analysis.
While six scAAV9-treated dKO mice were sacrificed at 12 weeks of age for muscle analysis, six others were kept for long-term analysis to investigate the effect of exon skipping on lifespan.
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Twenty fish were used for the liver analysis and 18 fish for the muscle analysis.
Serial 4-μm sections were taken through the entire head (for analysis of the brain) or thorax (for indirect flight muscle analysis).
A striploin steak was obtained from each carcass at 48 h post harvest for muscle Ca analysis and Warner-Bratzler shear force (WBSF) determination.
For muscle fiber area analysis, gastrocnemius muscle tissue was processed without PFA fixation to avoid shrinkage.
As a result of these exclusions, the final cross-sectional study population for muscle strength and TUGT analysis were composed of 1,131 subjects [age 68.95 ± 6.94 years (mean ± standard deviation, SD); males: 47.39 %], and for 4-m gait speed analysis was composed of 938 subjects.
Muscle sections were placed in a skinning solution 28 in preparation for single muscle fiber analysis or placed in RNAlater® (Life Technologies Inc ,Carlsbad, CA) and stored at −20°C until the mRNA IGF-1 and IGFBP-5 analysis was performed.
Given that the data had to be grouped by muscle for analysis, the groups are heterogeneous, the sample size relatively small and thus interpretation limited.
Stimulus pulses were generated by pClamp 10 software (Molecular Devices, Sunnyvale, CA), and the applied currents were 6 μA ± 3 with fixed stimulus duration at 0.3 ms. 30 evoked EJPs were recorded from each muscle for analysis.
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