Biotinylated antibodies

The MOG-specific response of T cells isolated from immunized mice either exposed to hypoxia or normoxia was analyzed for cytokine-secreting phenotype by ELISPOT assay. Briefly, splenocytes (2 × 10⁵ cells/well) were plated into IL-17A or IFN-γ-coated 96-well filtration plates (Millipore) and stimulated with MOG peptide overnight at 37 °C in humidified 5 % CO₂/air incubators. After 24 h, the plates were incubated with the appropriate biotinylated antibodies (all antibodies from ebiosciences), and the cytokine-producing cells were visualized with streptavidin-alkaline phosphatase system (Vector Laboratories), and the plates were analyzed using the CTL ImmunoSpot Analyzer (Cellular Technology Limited, Shaker Heights, OH) with ImmunoSpot software. The frequency of cytokine-producing cells was expressed as the difference between the mean number of spots and the mean background for each experiment. The data were presented as the mean ± SEM of each group, n = 3 mice, and performed in triplicate.

To analyse the cellular localization of TLR4, NF-κB/p65 and IL-6, immunohistochemical staining was performed on samples from three patients at room temperature using the avidin-biotinylated peroxidase complex (ABC) technique. To preserve the outer membranes of the CSDH samples, the samples were incubated in 10 mL of ice-cold 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 3 h. Serial axial cryostat sections (10 μm) were placed on slides for staining. Non-specific immunoreactivity was blocked by incubation with goat serum for 30 min. The samples were treated with primary antibodies against TLR4 (#sc-293072, Santa Cruz Biotechnology) diluted 1:20, NF-κB/p65 (#8242, Cell Signaling Technology) diluted 1:500 and interleukin-6 (IL-6; #12153, Cell Signaling Technology) diluted 1:50 overnight at 4°C. After being washed, the samples were incubated with biotinylated anti-mouse or rabbit IgG for 1 h and then ABC for 1 h. Sera used for the blocking step, biotinylated antibodies, and ABC were purchased from Vector Laboratories (Burlingame, CA). The reaction products were developed by incubating the sections in 0.05% 3,3'-diaminobenzidine tetrachloride and 0.01% H₂O₂ in 50 mM Tris-HCl (pH 7.5) for 10 min. The histopathological analyses without these antibodies were used as negative controls.