T1175

Animals were anesthetized, and perfused intracardially with 4% paraformaldehyde (PFA) in 0.01 M phosphate-buffered saline (PBS). Brains, spinal cords, and biceps brachii were collected for preparing sections with a sliding microtome (Leica, Germany). Floating methods were used for immunostaining. Briefly, after washing with 0.01 M PBS plus 0.3% Triton, sections were blocked in 3% bovine serum albumin plus 10% normal donkey serum for 2 h, and incubated with the primary antibodies overnight at 4 °C. The primary antibodies included goat anti-choline acetyltransferase (ChAT; 1:500, AB144p, Millipore), rabbit anti-protein kinase Cγ antibody (PKCγ; 1:400, ab109539, Abcam), rabbit anti-tyrosine hydroxylase (TH; 1:1000, AB152, Millipore), rabbit anti-serotonin (5-HT; 1:1500, S5545, sigma), rabbit anti-NF200 (1:500, n4142, Sigma), and guinea pig anti-vGlut2 (1:1,000, AB2251, Millipore). After three rinses in 0.01 M PBS, signal was disclosed by secondary fluorescent antibodies (Alexa Fluor 488 or 546, 1:1,000, A11073/A11055/A21206/A10040, Thermo Fisher), and α-bungarotoxin conjugated to Alexa Fluor 546 (α-BT; 1:1,000, T1175, Molecular Probes) was used to label acetylcholine receptors.

Five months after surgery, forty µm-thick frozen longitudinal sections of the biceps brachii were prepared using a sliding microtome (Leica SM 2010R). Rabbit anti-neurofilament 200 (NF200, 1∶1000, N4142, Sigma) and alpha-bungarotoxin conjugated to Alexa fluor 546 (a-BT, 1∶1000, T1175, Molecular Probes) were used to label axons and acetylcholine receptors (AchRs). For each section, five isolated AchR clusters were photographed under100× oil objective and 100 NMJs were studied in 4 animals in each group. NMJs were ranked in three categories: mature, denervated and immature (remodeled and neoformed) as described [27] (link), and the proportion of each type was calculated.

At the end of the 12-week experimental period, mice were deeply anesthetized with 4% tribromoethanol and perfused intracardially with 4% paraformaldehyde in 0.01 M phosphate-buffered saline (PBS, pH 7.4). Spinal C5–C7 segments and biceps (six animals in each group) were collected and post-fixed in the same fixative overnight, and then immersed in 10%-20%-30% sucrose until they sank. Ten micrometer transverse spinal sections and 40-μm horizontal biceps sections were prepared with a sliding microtome (Leica). Sections were kept in 12-well plates at 4°C. After washing with PBS, sections were blocked in 10% goat serum plus 3% bovine serum albumin for 2 h, and incubated with the primary antibodies overnight at 4°C. The primary antibodies included: goat anti-choline acetyltransferase (anti-ChAT; 1:500; AB144p, Millipore), rabbit anti-oligodendrocyte transcription factor 2 (anti-Olig2; 1:2,000, AB9610, Millipore), mouse anti-serotonin (anti-5-HT; 1:500, ab6336, Abcam) and rabbit anti-tyrosine hydroxylase (anti-TH; 1:500; AB152, Millipore). Sections were rinsed in 0.01 M PBS and incubated with the secondary fluorescent antibodies (Alexa Fluor 488 or 546; 1:1,000, A21202/A21206/A11055, Thermo Fisher) for 2 h. α-bungarotoxin conjugated to Alexa Fluor 546 (α-BT; 1:1,000, T1175, Molecular Probes) was used to label acetylcholine receptor clusters.