Rabbit anti mouse igg peroxidase conjugate

The primary antibody used for all Western blots in this study was ANTI-FLAG M2® Monoclonal Antibody, mouse-purified IgG (Sigma) at a final concentration of 10 μg/ ml and the secondary antibody used was Rabbit Anti-Mouse IgG, Peroxidase Conjugate (Sigma) at a dilution of 1:25 000–40 000. Membranes were incubated with the primary antibody for 1 hr at room temperature or at 4°C overnight with gentle agitation. This was followed by three washes in TBST (Tris-Buffered Saline and Tween 20) for 5 min each. Incubations with the secondary antibody were carried out for 1 hr at room temperature with gentle shaking. This was followed by five wash steps of 5 min each with TBST at room temperature. Chemiluminescent Peroxidase Substrate (CPS) Reagent (Sigma) was then added and the membrane was exposed to X-ray film.

Newcastle disease virus-based recombinant influenza vaccine (rL–H5) and H5 subtype avian influenza virus antigen (H5–Ag) were purchased from Harbin Weike Biotechnology Development Company, Heilongjiang, China. OVA, concanavalin A (Con A), lipopolysaccharide (LPS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), RPMI-1640 medium, and rabbit anti-mouse IgG peroxidase conjugate were purchased from Sigma Chemical Co., Saint Louis, MO, USA; goat anti-mouse IgG1, IgG2a, and IgG2b peroxidase conjugate were from Southern Biotech. Assoc., Birmingham, AL, USA; cytokine (IL-2, IL-10, and IFN-γ) detecting ELISA kits were from Wuhan Boster Biological Technology Co. Ltd., Hubei, China. Quil A was kindly provided by Brenntag Nordic A/S, Denmark. Fetal calf serum (FCS) was purchased from Hyclone, Utah, USA.
Human leukemia K562 cells, sensitive to natural killer (NK) cells, were purchased from Institute of Cell Biology, Chinese Academy Sciences. They were maintained in the logarithmic phase of growth in RPMI-1640 medium supplemented with 2 mM L-glutamine, 100 IU/ml penicillin, 100 μg/ml streptomycin, and 10 % FCS at 37 °C under humidified air with 5 % CO₂.

Newcastle disease virus (NDV)-based recombinant influenza vaccine (rL-H5) and H5 subtype AIV hemagglutination inhibition detecting antigen (H5Ag) were purchased from the Harbin Weike Biotechnology Development Co., Heilongjiang, China. RPMI medium was from Hyclone/GE Healthcare, Logan, UT, USA; fetal bovine serum (FBS) was from Gibco, Grand Island, NY, USA. Rabbit anti-mouse IgG peroxidase conjugate were purchased from Sigma Chemical Co., St. Louis, MO, USA; goat anti-mouse IgG1 and IgG2b peroxidase conjugates were from Southern Biotech. Assoc., Birmingham, AL, USA; goat anti-mouse IgG2a peroxidase conjugates were from Abcam, Cambridge, UK. Trizol reagent was purchased from Invitrogen, Carlsbad, CA, USA; revert Aid™ M-MuLV reverse transcriptase was from Fermentas, USA; diethylpyrocarbonate (DEPC), ribonuclease inhibitor, and oligo(dT)₁₈ were from Shanghai Sangon Biological Engineering Technology Co., Ltd., Shanghai, China; FastStart Universal SYBR Green Master (ROX) was from Roche Diagnostics Ltd., Shanghai, China. Agilent 4 × 44 k whole mouse genome microarray was provided from Agilent Technologies. Santa Clara, CA, USA.