Ab1987

Dorsal roots and L4 DRG (n = 4 per group) were fixed in 4% paraformaldehyde (PFA) overnight before being transferred to the 30% sucrose cryoprotection solution, where they stayed overnight at 4 °C. Tissue was then embedded in Tissue-Tek (Sakura) and snap frozen in isopentane on dry ice. Longitudinal sections were cut at 10 μm in a cryostat. After drying, tissue sections were incubated in 20% methanol for 5 min, blocked with a buffer containing 10% fetal bovine serum in 0.3% Triton X-100 in PBS for 1 h at room temperature, followed by overnight incubation with the primary antibodies against mouse mAb pSer129-α-Syn (11A5, 1:5,000) and neurofilament M (NF-M) (Millipore AB1987, 1:200) at 4 °C. Alexa Fluor 488 anti-mouse and Alexa Fluor 568 anti-rabbit-labelled secondary antibodies (Molecular Probes, A10037 and A21206 respectively, 1:500) were incubated for 2 h and Hoechst (Sigma, 1:10,000) was used to label the nuclei, visualized in blue. Slides were mounted with DAKO fluorescent mounting medium and Z-stacked images captured by confocal microscopy (LSM780, Carl Zeiss, Germany).

For western blot analysis, we used an anti-SMN mouse monoclonal antibody (BD Transd Lab, clone 8, #610646; 1:10,000), an anti-Tubulin mouse monoclonal antibody (Sigma, clone DM1A, #T9026; 1:50,000), and a HRP conjugated goat anti-mouse secondary antibody (Jackson #115-035-044; 1:10,000). For spinal cord immunohistochemistry, we used goat anti-ChAT (Millipore #AB144P; 1:100), guinea pig anti-VGluT1 (Covance, custom made; 1:5,000) [18 (link)], rabbit anti-p53 (Leica Novocastra #NCL-p53-CM5p; 1:1,000) and rabbit anti-phosporylated-p53^S15 (Cell Signaling #9284, Lot: #15; 1:250) as primary antibodies. For muscle immunohistochemistry, we used guinea pig anti-Synaptophysin 1 (Synaptic Systems #101–004; 1:500), rabbit anti-Neurofilament M (Millipore #AB1987; 1:500), and Alexa Fluor™ 555 conjugated α-bungarotoxin (Invitrogen, #B35451; 1:500). Species-specific secondary antibodies coupled to Cy3 or Cy5 were used as appropriate (Jackson ImmunoResearch Laboratories, Inc; 1:250).

The following antibodies and relative dilutions (in brackets) were used in this study: anti-Neurofilaments light (NfL; 1:1000) (clone EP675Y, Rabbit, Millipore), anti-Neurofilament Medium (NfM; 1:1000) (AB1987, Rabbit, Millipore), anti-Neurofilament heavy (NfH; 1:1000) (N4142, Rabbit, Sigma-Aldrich), Swine Anti-Rabbit Immunoglobulins (1:50,000) (P021702-2, DAKO).