Horseradish peroxidase conjugated anti mouse igg

The titers of IgE, IgM, and IgG1 in culture medium of B cells were measured by using ELISA (BD Pharmingen, San Diego, CA, USA). Levels of C3 and haptoglobin in CM were measured by using the C3 ELISA kit (Kamiya Biomedical Co.) and the Haptoglobin ELISA kit (Abnova, Taipei, Taiwan). TCTP and cathepsin L in the CMs were detected by performing a western blot analysis using the following antibodies; purified mouse anti-TCTP (BD Biosciences, San Jose, CA, USA), mouse anti-cathepsin L (Pierce, Rockford, IL, USA), and horseradish peroxidase-conjugated anti-mouse IgG (Santa Cruz Biotechnology, Dallas, TX, USA).

After treatment with rutin for 3 days, rats were scarified and approximately 15 cm long spinal cord containing T₈ and T₉ were taken out for further testing. 10-mg spinal cord tissue samples were incubated with 100 μL tissue lysis buffer (Beyotime) for 30 minutes on ice. Homogenates were centrifuged at 12,000 × g for 10 minutes at 4°C. The supernatants were collected, and the protein concentration was determined using a bicinchoninic acid kit (KeyGen Biotech). Equal amounts of protein were resolved on 12% sodium dodecyl sulfate-polyacrylamide gel electropheresis gels and then transferred to polyvinylidene fluoride membranes (0.22 μm). Membranes were blocked with PBS containing 5% non-fat milk to block nonspecific binding. Then, the membranes were incubated with anti-p38 MAPK mouse monoclonal antibody (1:2,000; sc-398305, Santa Cruz Biotechnology, Dallas, TX, USA) and anti-β-actin rabbit polyclonal antibody (1:500; D110007, Sangon Biotech, Shanghai, China) overnight at 4°C, followed by incubation with horseradish peroxidase-conjugated anti-mouse IgG (1:1,000; sc-2005 and sc-2004, Santa Cruz Biotechnology) for 2 hours at room temperature (24–26°C). Signals were quantified using the Gel Doc XR+ Gel Documentation System (Bio-Rad, Hercules, CA, USA). Grayscale value (/internal reference) was detected by the image J software (NIH, Bethesda, Maryland, USA).