Goat anti rabbit dylight 488

Rats were deeply anesthetized with a lethal dose of pentobarbital (80 mg/kg, i.p.) and transcardially perfused with 0.01 M PBS followed by 4% paraformaldehyde in 0.1M PBS for 20 min. The spinal cords were removed, left in fixative for 24 hours at 4°C, cryoprotected in 30% sucrose in 0.1 M PBS and stored at 4°C. Frozen segments were sectioned (25 µm) in the sagittal plane on a Leica cryostat. Prior to immunostaining, sections were incubated in 0.01 M PBS with 0.2% Triton X-100 (PBS-T) and blocked with 5% normal goat serum for 1 hour at RT. Following an overnight incubation at 4°C with primary antibodies, sections were washed 3 times with PBS-T (3–5 min) and incubated with DyLight 488 goat anti-rabbit (1∶500), DyLight 594 goat anti-mouse (1∶500; Jackson Immuno Research, Suffolk, UK) or Alexa Fluor 488 goat anti-rabbit (1∶500) and Alexa Fluor 594 goat anti-mouse (1∶500, Alexa Fluor, Life Technologies Corporation (Molecular Probes), Carlsbad, CA, USA) antibodies for 45 min at RT. The reaction was terminated by washes (3 times with PBS-T; 3–5 min), followed by 0.01 M PBS. Next, sections were immersed for 5 min in 0.2 µM bisbenzimide H 33258 (Hoechst; Sigma-Aldrich) to stain nuclei, mounted in Mowiol (Mowiol 4–88, Sigma-Aldrich (Fluka), St. Louis, MO, USA), coverslipped, and kept in the dark at 4°C until analysis.