The rats were anesthetized deeply by intraperitoneal injection of sodium pentobarbital (90 to 120 mg/kg) and transcardially perfused with ice-cold phosphate-buffered saline (PBS) and 4% paraformaldehyde. The spinal cords were postfixed for 4 hours in 4% paraformaldehyde and cryoprotected overnight at 4°C in PBS containing 30% sucrose (27). The frozen tissues were sectioned (14-μm thickness) with a cryostat at −20°C. After blocking overnight in PBS containing 10% normal goat serum (NGS) and 0.25% Triton X-100, the sections were incubated with rabbit anti–Cbl-b antibody (Proteintech, Rosemont, IL, USA) and mouse anti-NeuN antibody (Millipore, Temecula, CA, USA), mouse anti-CD11B antibody (OX-42; Bio-Rad, Hercules, CA, USA), or mouse anti-GFAP antibody (Sigma-Aldrich) overnight at 4°C. The sections from mice receiving viral injection were incubated with mouse anti-GFP antibody and rabbit anti-Lmx1b, rabbit anti-Pax2, or rabbit anti-vAchT antibody (Proteintech). After three washes with 10% NGS in PBS, the sections were incubated with Alexa Fluor 488–conjugated goat anti-mouse and Cy3-conjugated goat anti-rabbit secondary antibodies at room temperature for 2 hours before image capture. The experiments were conducted and analyzed blindly.

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