Rabbit anti occludin polyclonal antibody

Rabbits were anesthetized with an overdose of pentobarbital, and the L_4–6 segments were rapidly collected for western blot analysis 48 hours after surgery to measure the expression levels of MMP-9, NF-κB (p65), claudin-5 and occludin. Briefly, the spinal cord tissues were homogenized, and total proteins were purified using cell and tissue protein extraction reagents according to the manufacturer's instructions (Wuhan GuGe, Wuhan, Hubei Province, China). The samples were subjected to SDS-PAGE, and the proteins were blotted onto a membrane and probed with the following antibodies: mouse anti-NF-κB (p65) polyclonal antibody (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-MMP-9 polyclonal antibody (1:1,000; Affinity, Zhenjiang, Jiangsu, China), goat anti-claudin-5 polyclonal antibody (1:500; Santa Cruz Biotechnology), rabbit anti-occludin polyclonal antibody (1:1,000; Abcam, Cambridge, UK) and horseradish peroxidase (HRP)-conjugated secondary antibodies (goat and donkey; 1:3,000; Wuhan GuGe). The membranes were incubated with primary antibodies overnight at 4°C and with secondary antibodies for 30 minutes at room temperature, blocked and visualized by enhanced chemiluminescence. Semi-quantitation of scanned films was performed using Quantity One software (Bio-Rad, Hercules, CA, USA).