Abs787

Tissue from mice was dissected and fixed in 4% paraformaldehyde in PBS for 4 hours for spinal cords, and 1 hour for sciatic nerves at 4°C before being transferred into 30% sucrose solution over-night at 4°C for cryoprotection. Tissue was embedded in OCT (1:1 OCT and 30% sucrose for sciatic nerve) and using a cryostat, where spinal cord (25 μM) and sciatic nerve (10 μM) sections were collected on polysine-coated slides (Thermo Scientific). For immunohistochemistry, sections were permeabilized in 0.3% Triton X-100 in PBS and then blocked (4% BSA, 0.3% Triton X-100 in PBS) for 30 minutes at room temperature before overnight incubation with primary antibody solution at 4°C: anti-PGK1 (Millipore, ABS787) and mouse anti-S100 (Abcam, AB7852) or mouse anti-NF heavily phosphorylated 200 (convence, SMI-31R). After PBS washes, sections were incubated with secondary antibody solution for 2 hours at room temperature: Alexa Fluor 488 donkey anti-rabbit IgG (1:500, Life Technologies, A-21206), Alexa Fluor 594 donkey anti-mouse IgG (1:500, Life Technologies A21203). Sections were counterstained with DAPI nuclei stain (1:1,000, Life Technologies, D1306) for 10 minutes before being mounted with 10% Mowiol (Polysciences). Images were taken using a Zeiss LSMZ10 confocal microscope.

All protein (zebrafish embryos and mouse tissue) was extracted in RIPA buffer (ThermoScientific) with 1% protease inhibitor cocktail (Sigma) and homogenised. Protein concentration was determined by BCA assay (ThermoScientific). SDS-page was performed using 20% pre-cast NuPage 4–12% BisTris gradient gels (Life Technologies) and transferred to PVDF membranes by the iBlot 7 minute semi-dry blotting system (Life Technologies). Following blocking, membranes were incubated overnight at 4°C using mouse anti-SMN (1:1,000, BD biosciences, 610153), rabbit anti-PGK1 (1:1000, Millipore, ABS787), rabbit anti-ATP5A (1:1000 ab176569, Abcam), rabbit anti-cyt C (1:1000, Abcam, ab18738) mouse anti-COX IV (1:1000 Abcam, ab14744), mouse anti- GAPDH (1:1000, Abcam, ab9484,). After PBS washes, secondary antibodies were added for 1 hour at room temperature. Secondary antibodies used were; IR dye 800CW goat anti-mouse IgG (926–32210), IR dye 800CW goat anti-rabbit IgG (926–3211), IR dye 680RD goat anti-mouse IgG (926–68070), IR dye 680RD goat anti-rabbit IgG (926–68071), all 1:5,000, LI-COR Biosciences. Membranes were imaged using an odyssey infrared imaging system (Li-COR, Biosciences) as described previously [80 (link)], and quantified using an image studio software (Li-COR). To determine final relative protein expression, band intensities were normalized to a loading control or a ponceau stain.