Anti sv2a

Prior to dissection, mice were euthanized with 10% Nembutal and subsequent cervical dislocation. The gastrocnemius muscle was dissected, snap-frozen in liquid-nitrogen-cooled isopentane and stored at −80°C. On the day of the experiment, longitudinal cryosections of 20 μm were fixed in 4% PFA for 10 min before rinsing with 1X PBS. Tissue was subsequently permeabilized with PBS-T for 5 min and blocked in PBS-T supplemented with 10% normal donkey serum (NDS, Sigma-Aldrich) for 1 h. Neuronal axons (anti-NFL light chain antibody (Alexa Fluor 488 conjugated, Cell Signaling, 8024S, 1/500), presynaptic terminals (anti-SV2A, Developmental Studies Hybridoma Bank, 1/100), and motor endplates (Alexa Fluor 555-conjugated α-bungarotoxin, Life Technologies, B35451, 1/5,000) were stained with primary antibodies dissolved in PBS-T supplemented with 10% NDS and incubated overnight. Slides were mounted with Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA) and images were acquired with a Leica TCS SP8 confocal laser scanning microscope (Leica Microsystems). For each experimental condition, at least 100 NMJs were analyzed for innervation on a Zeiss Imager M1 microscope system using a 40X objective (Carl Zeiss, AG). Assessments were undertaken by an investigator blinded to the different groups.

Prior to dissection, the mice were euthanized by CO₂ inhalation and subsequent cervical dislocation. The gastrocnemius muscle was dissected and snap-frozen in liquid nitrogen-cooled isopentane. Cryosections (20 µm) were fixed with 4% PFA during 10 min and rinsed with PBS. The tissue was permeabilized using PBS-1% Triton™ X-100 for 30 min. Non-specific binding was blocked using 5% normal donkey serum (Sigma-Aldrich) dissolved in PBS-0.3% Triton™ X-100. Antibodies, visualizing nerve axons [anti-neurofilament light chain antibody (Alexa Fluor® 488 conjugated, Cell Signaling, 8024S, 1/500)], the presynapse (anti-SV2A, Developmental Studies Hybridoma Bank, 1/100) and the neuromuscular endplate [α-bungarotoxin (Alexa Fluor®555-conjugated, Life Technologies, B35451, 1/5000)] were dissolved in blocking solution and incubated overnight. Representative confocal images were obtained on a Leica TCS SP8 confocal laser scanning microscope (Leica Microsystems). A minimum of 100 neuromuscular junctions were analysed for innervation per biopsy and samples of n = 3 mice were used per treatment condition. Z-stacks were acquired using the Leica Application Suite X software (LAS X 3.3.0, Leica Microsystems) and maximum intensity projections images were generated in Fiji (NIH) from 10 plane Z-stacks through a 20-μm thick section. All analyses were performed in a blinded manner.