Postmortem Analysis of SOD1-Mutated ALS

Postmortem lumbar spinal cords from two patients with neuropathologically-proven SOD1 mutated ALS (mean age 52.3 years), five patients with sporadic ALS (mean age 67.4 years), and five age-matched control patients (mean age 67.2 years) were analyzed. Control cases included three patients with other neurodegenerative diseases (one with Parkinson’s disease; one with dentatorubropallidoluysian atrophy, one with spinocerebellar ataxia), and two without neurodegenerative diseases (one with cerebral infarction; one with epilepsy) (Table S1). During the autopsy, spinal cords were removed, and blocks of the lumbar levels of the spinal cords were immediately placed in 10% buffered formalin and embedded in paraffin. No pathological changes were noted in the spinal cords from control patients. For immunohistochemistry, after heat retrieval by autoclaving (10 min at 121°C in 10 mM sodium citrate buffer), 6-μm-thick sections were incubated overnight at 4°C with D3-1 diluted 1:1,000 with PBS containing 3% BSA (PBS-BSA). Bound primary antibodies were detected with a Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA) with 3,3′-diaminobenzidine tetrahydrochloride used as the chromogen. The staining specificity was confirmed by replacing the primary antibody with the appropriate amount of PBS-BSA. No reaction product deposits were seen in these control-stained sections. Written informed consent was obtained from all individuals or their guardians before the analysis. The protocols for human sample experiments (R1038) were approved by and performed under the guidelines of the Kyoto University Graduate School of Medicine Ethics Committee. Informed consent was obtained from all individuals or their guardians before the analysis.