Rabbit anti phospho nf κb

Rats were anesthetized by 10% chloral hydrate (300-350 mg/kg, intraperitoneal injection) and perfused transcardially with 100 mL phosphate-buffered saline (0.01 M PBS, pH 7.4), followed by 200 mL ice-cold 4% paraformaldehyde in 0.2 M phosphate buffer (300 mL, pH 7.4). The L4-6 spinal cord tissues from rats were removed immediately and post-fixed for 4-6 h at 4°C, and then cryoprotected by immersion for 24-48 h in sucrose gradients (5%, 10%, 15%, 20% and 30%) with 0.01 M PBS at 4°C. The spinal cord tissues were embedded with OCT, at -20°C and sectioned on a cryostat (Leica CM1900) at 30 µm in the transverse plane. The frozen sections were collected in PBS. After 3 washes in PBS, sections were incubated in PBS with 0.3% Triton X-100 (PBST) for 48 h at 4°C and then incubated with primary antibodies of rabbit anti-phospho-JNK (1:100 dilution; Cell Signaling Technology) or rabbit anti-phospho-NF-κB (1:100 dilution; Cell Signaling Technology). After rinsing in PBS, sections were incubated with the second antibodies solution in the darkness for 2 h at room temperature. Finally, sections were rinsed, mounted and cover-slipped with glycerol containing 2.5% anti-fading agent 1,4-di-aza-bi-cyclo-2,2,2-octane (DABCO, Sigma). Tissue sections were examined using laser scanning confocal microscopy (TCS SP2, Leica). ImageJ (NIH) was used for image quantification and analysis.