Goat anti rabbit immunoglobulin g igg

We separated 35 μg of protein by SDS-PAGE. After transfer to a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA), membranes were blocked with 5% nonfat dry milk in 0.1% Tween-PBS (TBST) and incubated overnight at 4°C with primary antibody in 5% bovine serum albumin/TBST [mouse anti-p53 (Cell Signaling, Danvers, MA, USA), mouse anti-cytochrome c (BD Biosciences, Franklin Lakes, NJ, USA), rabbit anti-cleaved caspase 3 (Cell Signaling), mouse anti-HSP 70 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-cytochrome c oxidoreductase (COX-IV) (Invitrogen), mouse anti-histone H1b (Abcam, Cambridge, MA, USA)] followed by horseradish peroxidase (HRP)-conjugated secondary antibodies (goat anti-mouse IgG or goat anti-rabbit immunoglobulin G (IgG; Jackson ImmunoResearch, West Grove, PA, USA) (in 5% nonfat dry milk/TBST). HRP-conjugated mouse anti-β-actin (Sigma-Aldrich) was used for normalization of cytosolic samples. Blots were revealed using enhanced chemiluminescence Amersham ECL Western Blotting Detection Reagent (GE Healthcare, Little Chalfont, Buckinghamshire, UK) and visualized, captured, and analyzed using a LAS system and Image Quant software (GE Healthcare).

Rats in the SCI and UCZN +980 groups were executed at a certain point in time (just after SCI and 4 weeks after injury). The spinal cord was separated and sectioned longitudinally. The sections were baked at 60°C for 1 hour and immersed into xylene and ethanol for dewaxing. Part of the sections was stained in H&E after washing with tap water or PBS. Other sections were immersed in sodium citrate buffer solution (0.01 M, pH 6.0) at high pressure for 5 min to repair the antigen, followed by cooling for 40 min. After inactivating peroxidase (in 3% H₂O₂ solution for 15 min), the sections were incubated overnight at 4°C with primary antibodies. The primary antibodies included IL-1 (rabbit, bs-0812R, 1:1000), IL-6 (rabbit, bs-0782R, 1:600), and TNF-α (rabbit, bs-2081R, 1:500). The sections were incubated for 30 min at room temperature with a secondary antibody [goat anti-rabbit immunoglobulin G (IgG), 1:100, Jackson ImmunoResearch Laboratories]. The nuclei were restained by hematoxylin. After that, all slices were immersed into clean xylene transparent for 5 min and sealed by neutral gum. The sections were observed with a 40-fold objective lens to determine the growth status. The lesion areas in the visual field were calculated with ImageJ (National Institutes of Health, USA).