Rhodamine red conjugated bungarotoxin

Gactrocnemius muscle was excised, dissected into smaller sections, and fixed in 4% paraformaldehyde, and then incubated with 1 μg/mL Rhodamine Red-Conjugated Bungarotoxin (Sigma-Aldrich). Tissues were then treated with methanol at −20 °C for 5 min, and afterwards blocked in blocking solution (1 mg/mL BSA, 0.1% triton in PBS) for 1 hour. Tissues were then rocked with appropriate primary antibodies diluted in blocking solution at room temperature overnight. Antibodies were used at the following concentrations: NFH 1:500 (Chicken; Abcam, ab72996); Elavl2 1:50 (Rabbit; Proteintech 14008-1-AP); Synaptophysin 1:500 (Mouse; millipore, mab5258) After washing, secondary antibodies (DyLight 405 anti-chicken 1:500; AlexaFluor 488 anti-Rabbit 1:500; AlexaFluor 647 anti-mouse 1:500) were added for 4 hours at room temperature. Muscle fibers were spread into monolayers under a stereomicroscope and affixed to slides using VectaShield (Vector Laboratories).

Gastrocnemius was excised from adult mice and cleared of connective tissue, washed in PBS, fixed in 4% PFA, washed again, and finally incubated with 1 μg/mL Rhodamine Red-conjugated Bungarotoxin (Sigma). Tissues were washed, and then treated with MeOH at −20°C for 5 min, and then washed and blocked in blocking solution for 1 h. Next, the tissues were rocked with appropriate primary antibodies diluted in blocking solution at room temperature overnight. After having been washed, secondary antibodies were added for 4 h at room temperature. Muscle fibers were spread into monolayers under a stereomicroscope and affixed to slides using VectaShield (Vector Laboratories). Cover slides were sealed with clear nail polish.

GC was excised from P60 mice and cleared of connective tissue, washed in PBS, fixed in 4% PFA, washed once more, and then incubated with 1 g/ml Rhodamine Red-Conjugated Bungarotoxin (Sigma-Aldrich). Tissues were washed and then treated with methanol at −20°C for 5 min, washed, and then blocked in blocking solution for 1 h. Tissues were then rocked with appropriate primary antibodies diluted in blocking solution at room temperature overnight. Antibodies were used at the following concentrations: anti-Neurofilament Heavy Chain 1:500 (NFH, Abcam, ab72996; 1:1000), synaptophysin (Millipore, MAB5258; 1:300), synaptotagmin (Alomone Labs, ant-003; 1:300), anti-NRP1 (1:100), anti-Sema3A (1:100), anti-NRP2 (1:100), and anti-Sema3B (1:100). After having been washed, secondary antibodies (DyLight 405 anti-chicken 1:500; AlexaFluor-488 anti-chicken 1:500; AlexaFluor-647 anti-rabbit 1:500) were added for 4 h at room temperature. Muscle fibers were spread into monolayers under a stereomicroscope and affixed to slides using VectaShield (Vector Laboratories). Cover slides were sealed with clear nail polish.