Goat anti rabbit igg555

All rats were then deeply anesthetized with 1% pentobarbital sodium (50 mg/kg, intraperitoneal) and transcardially perfused with normal saline containing 0.002% NaNO₂ and 0.002% heparin, followed by a fixative of 4% paraformaldehyde in 0.1 M PBS (pH, 7.4). The spinal cord was dissected and post-fixed for 24 hours in the same fixative and kept in 30% phosphate-buffered sucrose at 4°C for 48 hours, then frozen and embedded in optimal cutting temperature compound. Successive T8–12 spinal cord segments were cut into longitudinal 25-μm sections. These were then washed with 0.01 M PBS for 5 minutes and blocked with 10% goat serum for 30 minutes at 37°C. The tissue sections were then incubated with primary antibodies diluted with 0.01 M PBS containing 0.3% Triton X-100 at 4°C overnight. After this, they were washed with PBS 3 times for 5 minutes, then incubated with secondary antibodies for 1 hour at 37°C. Cell nuclei were marked with the Hoechst3342 (Hoe). After 3 rinses, the slides were observed with a confocal microscope (Carl Zeiss, Oberkochen, Germany). Primary antibodies included rabbit anti-neurofilament protein 200 (1:200; Sigma-Aldrich, St. Louis, MO, USA) and rabbit polyclonal anti-NeuN (1:500; Abcam, Cambridge, UK). Secondary antibodies included goat anti-rabbit IgG488 (1:1,000; Abcam) and goat anti-rabbit IgG555 (1:1,000; Abcam).