Alexa 594 conjugated dextran

After retrograde identification of M1 and the frontal premotor cortex (PM) as the areas that originate porcine corticospinal axons, anterograde neural tracers were injected in those areas in three additional animals. For this, a craniotomy was performed on the left side of the skull over the cruciate and the superior frontal gyri, removing the bone from the midline to about 3 cm lateral, and from 3.5 cm rostral to 0.5 cm caudal to the frontal-parietal suture. Two different tracers were injected to study the terminations of axons arising from each cortical region. Alexa 594-conjugated dextran (10,000 MW, Thermo Fisher Scientific Inc., D22913) was injected in M1, and Alexa 488-conjugated dextran (10,000 MW, Thermo Fisher Scientific Inc., D22910) was injected in PM. Both tracers were dissolved at 10% in saline solution. Twelve injections (4-μL each, at 2 mm and 4 mm of depth in the dorsoventral plane, separated 0.8-mm along the longitudinal axis of the respective cortical gyrus) of each tracer were made using a 50-μL Hamilton syringe mounted in a micromanipulator. The needle was set in place for 4 min to prevent reflux of the injected solution. The same total dose of 48 μL of each tracer was administered in all animals. The dura matter and scalp were closed separately.

After the embryos were decapitated and eviscerated, the target muscles and nerves were exposed in cold PBS, and Alexa594-conjugated Dextran (Cat# D22913, Thermo Fisher Scientific, USA) was injected using a picosplitzer (IM300, Narishige, Japan). The injected embryos were kept in oxygen-bubbled DMEM (Wako Pure Chemical, Japan) for up to 5 h at 30°C. After incubation, the embryos were fixed in 4% PA in 0.1 M PB for 5 h and then immersed in 20% sucrose in PBS overnight. The appropriate segments of the spinal cord were excised from the embryo and frozen-embedded in a mixture of 20% sucrose in PBS and Tissue-Tek embedding media (1:2 ratio; Sakura, Japan). Ten to fifteen muscles were injected with the tracer, and one-to six spinal cords with successful labeling were obtained for each muscle type.