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The lumbar enlargement of the spinal cord was dissected and subsequently submerged in phosphate-buffered 4% paraformaldehyde overnight at 4 °C. The specimens were dehydrated sequentially in 10%, 20% and 30% buffered sucrose solution before cryo-sectioned. The spinal cord was horizontally cut into 30-μm-thick serial sections on a cryostat. Every other sections were mounted on microscopic slides, and tracer-labeled motor neurons were visualized with an SP2 laser scanning confocal microscope (Leica Microsystems GmbH, Heidelberg, Germany), and manually counted.
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