Statistical Rapamycin in vitro analysis The specific statistical analysis used is noted below for each experimental approach. The Design Analysis Core at WFSM provided advice to the investigators, and the Design and Analysis Core performed select analyses. The number of animals used was based on experimental requirements for analysis and

were chosen for a two-sided analysis of population means with an acceptable probability of a Type I error (P-value) of 0.05 or less and a probability of a Type II error of 0.05 or less. Statistical significance occurred when Inhibitors,research,lifescience,medical power was determined to be 80% or better and the P-value was equal to or less than 0.05. NMJ innervation For counting innervated hind limb skeletal muscle NMJs, immunohistochemistry was performed on soleus and tibialis anterior (TA) muscles. Animals were transcardially perfused with 2% paraformaldehyde in 0.1 mol/L sodium phosphate buffer. The

muscles were dissected, rinsed twice with phosphate buffered saline (PBS), and placed Inhibitors,research,lifescience,medical in 20% sucrose for at least 72 h at 4°C. The muscles were embedded in 20% sucrose:OCT (2:1) and cut at 30 μm on the cryostat. Antigen retrieval was achieved using a sodium dodecyl sulfate pretreatment (Brown et al. 1996) and the sections Inhibitors,research,lifescience,medical were stained for the vesicular acetylcholine transporter (VAChT; Santa Cruz Biotechnology, Santa Cruz, CA) and neurofilament light chain (NF-L; Millipore, Billerica, MA). Alexa-fluor 488 secondary Inhibitors,research,lifescience,medical antibodies were used for detection (green). Sections were also labeled with Alexa-fluor-alpha-bungarotoxin (α-BTX; Invitrogen, Eugene, OR; red) (Maeda et al. 2004). NMJs that exhibited an overlap of red and green were considered innervated, while those that exhibited only α-BTX expression were considered denervated. All NMJs in every 30 μm section were analyzed. Inhibitors,research,lifescience,medical The percentage of innervated NMJs was determined in each treatment group using previously established counting criteria (Gifondorwa et al. 2007). Statistical differences

between WT and SOD1 groups were determined using unpaired t-test. MN and interneuron counts Briefly, mice were transcardially perfused with PBS followed by Bouin’s fixative. The lumbar region of the spinal cord was removed and embedded in paraffin. Twelve micrometer sections were cut and stained Thiamine-diphosphate kinase with a 5% thionin solution (Chu-Wang and Oppenheim 1978). Only healthy MNs were counted in every tenth section of the lumbar spinal cord using a well established reliable method that has been validated against an optical fractionator unbiased stereological counting method (Clarke and Oppenheim 1995). Healthy MNs are those that lie completely in the section with a nucleolus and normal MN morphology. Means and SEM were determined for each group. Statistical significance between the two groups was determined by a t-test with Bonferroni correction.