Immunohistochemistry, motor neuron and axon quantification

Tissue preparation for immunohistochemistry was described previously (Arnold et al., 2013). In brief, anesthetized mice were transcardialy perfused with phosphate buffered saline (PBS), followed by 4% paraformaldehyde (PFA) in phosphate buffer for fixation. L5 roots of 3 ~ 5 animals per genotype and age point were collected and incubated in 2% osmium tetroxide in 0.05 M cacodylate buffer. The roots were subsequently washed, dehydrated and embedded in Epon (Electron Microscopy Sciences) for sectioning. 1 μm-thick cross sections were stained with 1% toluidine blue for 30 s. Both motor and sensory axons from L5 roots were quantified as described (Arnold et al., 2013).

Brains and spinal cords were post-fixed in 4% PFA for 2 hr, cryoprotected in 30% sucrose for over 24 hr and embedded in Tissue-Tek. For immunohistochemistry cryosections (30 μm) of fixed spinal cord and brain were rinsed in PBS, incubated in a blocking solution containing PBS, 0.5% Tween-20, 1.5% BSA for 1 hr at room temperature and transferred for an overnight incubation at room temperature in PBS, 0.3% Triton-X100 supplemented with the following primary antibodies: mouse anti-HA (Covance) at 1:5,000, anti-p62/SQSTMQ (Enzo) at 1:500, goat anti-ChAT (Chemicon) at 1:300, rabbit anti-GFAP (Dako) at 1:1000 and mouse anti-Iba1 at 1:1000. Primary antibodies were washed with PBS and then detected using donkey anti-rabbit Cy3, anti-mouse Cy3, anti-goat Cy3 (1:500) coupled secondary antibodies (Jackson ImmunoResearch). The secondary antibodies were washed with PBS and the spinal cord sections were either directly mounted or further incubated with a monoclonal antibody against neuronal nuclei marker, NeuN-Alexa488 (1:1,000, Chemicon) for 1.5 hr at room temperature. The sections were washed with PBS and mounted. Analysis was performed on a Nikon Eclipse laser scanning confocal microscope.

All lumbar spinal cord choline acetyl-transferase (ChAT) positive motor neurons were counted in the ventral horn of at least 25 sections per animal (in three mice per genotype). The total number of motor neurons counted was then divided by the number of sections.

Evaluation of muscle innervation at the neuromuscular junction (NMJ) was performed by immunohistochemistry on gastrocnemius. Floating sections (40 μm) were incubated in a blocking solution containing PBS, 0.5% Tween-20, 1.5% BSA for 4 hr at room temperature and then in PBS, 0.3% Triton-X100 overnight at room temperature with the polyclonal rabbit anti-synaptophysin antibody at 1:50 (Invitrogen). The sections were washed with PBS and then incubated first with donkey anti-rabbit Cy3 (Jackson ImmunoResearch) and α-Bungarotoxin-Alexa488 (Invitrogen) at 1:500 for 1 hr at room temperature and then with FluoroMyelin red (Invitrogen) at 1:300 for 30 min. The sections were further washed with PBS and mounted. Analysis was performed on a Nikon Eclipse laser scanning confocal microscope. A total of approximately 1000 neuromuscular junctions were counted from at least 10 sections of gastrocnemius. Individual NMJs were considered as innervated when colocalization between synaptophysin and α-Bungarotoxin staining was over 20%.