CST axons were anterogradely labeled by a single injection of the CM-DiI dye (10 mg/mL in N, N-dimethylformamide) into the right-side motor cortex of P1 pups with the use of Hamilton micro syringe with 33 gage 30° needle. Pups were perfused at P3 with cold 1x PBS, brains with spinal cord were fixed in 4% PFA overnight, and 100μm sagittal cryosections were prepared along the anterior-posterior axis. They were mounted with Fluorogold anti-fade mounting medium then imaged under Keyence fluorescence microscope BZ-X700 with a Cy3 filter. Spinal cord CST axon length was measured based on the CM-DiI fluorescence signals from the superimposed images of individual mice (as shown in Fig. 5c ) after the pyramidal decussation (PD) by using the segment line tool in ImageJ.
Protocol detail
Tracing Corticospinal Tract Axons
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Axis
Axon
Brains
Cm dii
Cold
Cryosections
Dimethylformamide
Fluorescence
Fluorescence microscope
Fluorogold
Mice
Motor cortex
Needle
Pyramidal decussation
Spinal cord
Syringe
Corresponding Organization : Tufts University
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Variable analysis
independent variables
- Injection of CM-DiI dye (10 mg/mL in N, N-dimethylformamide) into the right-side motor cortex of P1 pups
- Spinal cord CST axon length based on the CM-DiI fluorescence signals from the superimposed images of individual mice after the pyramidal decussation (PD)
- Perfusion of pups at P3 with cold 1x PBS
- Fixation of brains with spinal cord in 4% PFA overnight
- Preparation of 100μm sagittal cryosections along the anterior-posterior axis
- Mounting of sections with Fluorogold anti-fade mounting medium
- Imaging under Keyence fluorescence microscope BZ-X700 with a Cy3 filter
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