Antibody titers were calculated by taking the inverse of the dilution at 50% saturation

Antibody titers were calculated by taking the inverse of the dilution at 50% saturation. 2.6. following coagulation and centrifugation (12,000?g, 10?min, RT) and stored at ?20C. 2.5. ELISA Screening of Immune Sera Aliquots of the antigen proteins were solubilized in 0.5% SDS and coated on 96-well MaxiSorp plates (Nunc, Roskilde, Denmark). After blocking, serum samples were applied to the plates in series dilution and incubated overnight at 4C. Bound IgG was detected with peroxidase conjugated secondary antibody (Santa Cruz, Dallas, TX) and quantified at OD450 following addition of Turbo TMB-ELISA substrate (Pierce). Antibody titers were calculated by taking the inverse of the dilution at 50% saturation. 2.6. Whole Brain Membrane Isolation and Solubilization One half of a freshly dissected rat brain was homogenized in 15?mL of 20?mM Tris-HCl, pH 8.0, containing protease inhibitors (mini Complete, Roche, Manheim, Germany) and centrifuged (800?g, 20?min, 4C) to remove whole cells and cellular debris. Following recentrifugation (54,000?g, 1?hr, 4C), the membrane pellet was washed and resuspended in solubilization buffer (20?mM Tris-HCl, 1% Triton X-100, 5?mM EGTA, 2?mM EDTA, 1?M NaCl, pH 8.0 containing protease Azaguanine-8 inhibitors) and incubated for 2?hr at 4C. Insoluble matter was pelleted (100,000?g, 30?min, 4C) and the supernatant was assayed for protein content using Biorad Protein Assay substrate (Biorad, Hercules, CA). 2.7. Antigen Capture ELISA 96-well MaxiSorp plates were coated with monoclonal NR1 antibody (mAB363; Chemicon, Temecula, CA) at 0.5?= 7) that Mouse monoclonal to EphB6 reached stage 4 or beyond (= 11) was randomly selected for TUNEL staining. In addition, one rat from the NR1[21C375] group and one rat from NR1[313C619] Azaguanine-8 group were not analyzed due to poor processing of the brain. Table 2 Seizure-induced hippocampal cell death. 0.05compared to either Homer1a or na?ve groups. 2.11. Immunoblot Analysis of Hippocampal Extracts Groups of rats were vaccinated as described above (= 5 per group) and sacrificed nine days after the boost injection. Brains were removed and the hippocampus was dissected. To obtain a crude lysate, tissue samples were sonicated in 10?mM Tris-HCl, 2?mM EDTA, pH 7.5, containing protease inhibitors (mini Complete, Roche). The lysates were centrifuged (800?g, 20?min, 4C) and the supernatants were Azaguanine-8 assayed for protein content. Aliquots were prepared for SDS-PAGE and stored C20C. Azaguanine-8 Twenty Azaguanine-8 0.05. 3. Results 3.1. Production and Confirmation of Antigen-Specific NR1 Antibodies NR1[21C375], NR1[313C619], and NR1[654C800] (Physique 1) were cloned into pET expression cassettes to generate recombinant protein with molecular size and purity assessed by SDS-PAGE (Physique 1(c)). Homer1a was chosen as a negative control antigen. All four proteins were used to vaccinate rats. ELISA screening of the sera against their respective antigens demonstrated a strong humoral response in all vaccinated rats that persisted for more than 4 months (Table 1(a)). Two complementary approaches were used to demonstrate that antigen-specific NR1 antibodies recognise and bind native NR1. Firstly, native NR1 from solubilized whole brain membrane was captured with immobilized NR1 monoclonal antibody and presented to immune sera in an antigen capture ELISA. OD450 signal showed that this three different NR1 antigens had generated antibodies with an affinity for the native protein with values for each serum correlating with its antigen-specific titer (Table 1(b)). The control Homer1a antisera did not recognise and bind to the captured native NR1. Secondly, IgGs from NR1[21C375], NR1[313C619], and NR1[654C800] rats were applied to na?ve hippocampal sections with CA1, CA3, and dentate gyrus immunoreactivity identical to that of an affinity-purified commercial NR1 polyclonal antibody (Determine 1(d)). IgGs from preimmune rat serum did not bind to the hippocampus. Homer1a antisera recognized native Homer protein by immunoblot screening against crude brain extract (not shown). Open in a separate window Physique 1 Generation of the NR1 fragments and screening of resultant vaccine antisera..