Mouse anti his monoclonal antibody

The strains and vectors used in this study are shown in Table S1. E. coli Top10 stored in our laboratory was used for all cloning steps. For recombinant proteins expression, pET-21a vector (Novagen, Madison, WI) was used with expression host E. coli BL21(DE3)pLysS (Novagen, Madison, WI). Restriction enzymes, Pyrobest DNA Ploymerase, dNTP, DNA Marker and gel extraction kits were obtained from TaKaRa (Dalian, China). The mouse anti-His monoclonal antibody and enhanced horseradish peroxidase (HRP)-Diaminobenzidine (DAB) chromogenic substrate kit were obtained from Tiangen (Beijing, China). FITC labeled donkey anti-mouse IgG, HRP labeled rabbit anti-mouse IgG, lead acetate, IPTG, and antibiotics were all purchased from Sangon Biotech (Shanghai, China). Pepsin (1:3000) was obtained from Amresco (OH, USA). Tryptone and yeast extract were obtained from Oxoid (Basingstoke, UK). E. coli was cultured in Luria-Bertani (LB) broth (1% tryptone, 0.5% yeast extract and 1% NaCl) supplemented with antimicrobial agents, as necessary.

Sma I, Taq DNA polymerase High Fidelity Kit, CIAP, T4 DNA ligase and protein marker were purchased from Takara (Osaka, Japan). Isopropyl-β-D-thiogalactopyranoside (IPTG) and imidazole were ordered from TaKaRa (Dalian, China). Superoxide dismutase activity assay kit (WST-1 method) was purchased from Jiancheng (Nanjing, China). Mouse anti-His monoclonal antibody and Horseradish peroxidase (HRP)-labeled goat anti-mouse IgG were from TIANGEN (Beijing, China) and Proteintech (Chicago, IL, USA), respectively. Enhanced chemiluminescence (ECL) and detection reagent were obtained from Biyuntian (Haimen, China). Ni–NTA resin and SYBR Green PCR kit were purchased from QIAGEN (Hilden, Germany) and Takara (Osaka, Japan), respectively. Bovine Cu/Zn-SOD was ordered from Biyuntian (Haimen, China). All other reagents were analytical grade or better and commercially available.

Immunofluorescence microscopy was performed as described in a previous publication [29 (link)]. Briefly, HKLs were prepared as described above and resuspended in PBS to a concentration of 10⁷ cells/ml. Then, rCsBAFF or rGST (10 μg/ml) was added to the cell suspensions. After incubation at 22°C for 1 h, the cells were centrifuged at 300 × g for 5 min. The cellular pellets were collected, washed with PBS, resuspended in PBS, and then incubated with mouse anti-His monoclonal antibody (dilution, 1/1000; Tiangen Biotech Co., Ltd.) and rat anti-IgM antibody (prepared above) at 22°C for 1 h. After incubation, the cells were centrifuged as above, resuspended in PBS, and then incubated with fluorescein isothiocyanate (FITC)-labeled goat anti-mouse IgG (dilution, 1/1000; Beijing Biosynthesis Biotechnology Co., Ltd., Beijing, China) and rhodamine B isothiocyanate-labeled goat anti-rat IgG (dilution, 1/1000; Beijing Biosynthesis Biotechnology Co., Ltd.) at 22°C for 1 h. The cells were collected by centrifugation, washed, re-suspended in PBS, and examined with a fluorescence microscope (E800; Nikon Corporation, Tokyo, Japan).