Goat anti mouse igg h l fitc

BHK-21 cells were seeded into a 6-well plate and cultured at 37 °C with 5% CO₂. A cell monolayer at 80% confluence was transfected with either H, optiH, H-IRES-N, optiH-IRES-optiN, optiH-IRES-IFN, or an empty plasmid vector, pIRES, using Lipofectamine 2000 (Thermo Fisher, Carlsbad, CA 92010, USA) according to the manufacturer’s instructions. At 48 h after transfection, the cells were fixed in 1 mL of 4% formaldehyde for 30 min at 37 °C and permeabilized in 1 mL of 0.5% Triton X-100 for 10 min. After three washes with phosphate-buffered saline (PBS), samples were blocked with goat serum for 30 min at 37 °C. The samples were washed and blocked after each subsequent antibody exposure to anti-CDV H protein monoclonal antibody (1C42H11 monoclonal antibody, VMRD, Pullman, WA, 99163, USA), anti-CDV N protein monoclonal antibody (CDV-NP monoclonal antibody, VMRD, USA), and anti-mink IFN-γ monoclonal antibody [19 (link)], as appropriate for the corresponding plasmid vectors, whereas goat anti-mouse IgG H-L (FITC) (Abcam, Cambridge 02139, UK) was used as the secondary antibody. Images were obtained using a fluorescence microscope (EVOS FL, Thermo, USA). Five fields of vision for each image were randomly selected, and the mean number of fluorescent cells was calculated and compared to evaluate protein expression levels.

Anti-IL-37 monoclonal antibody, Goat Anti-Mouse IgG H&L (FITC), and Mouse IgG1 were purchased from Abcam Corporation (Cambridge, UK). TWG (10 mg/tablet, Approval number Z35020431) was obtained in the form of tablets (Tripterygium wilfordii glycoside tablets) from Fujian Huitian Bio-pharma Co., Ltd. (GMP certificated, Fujian, China). The tablets were dissolved in the incubation medium at a final concentration of 5 mg mL⁻¹ as stock solution. Inhibitor of extracellular signal-regulated kinase 1 (ERK1)/ERK2, U0126, and inhibitor of p38 MAPK, SB 203580, were from Cell Signaling Technology (Beverly, MA, USA). RNAiso Plus, Prime Script™ RT Master Mix and SYBR® Premix Ex Taq (Perfect Real Time) were from Takara Biotechnology Co., Ltd. (Dalian, China). Primers were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). eBioscience Fixation/Permeabilization Concentrate and Permeabilization Buffer (10×) were from eBioscience Inc. (San Diego, CA, USA). Dimethyl sulfoxide (DMSO) and phorbol-12-myristate 13-acetate (PMA) were from Sigma (St. Louis. MO, USA). THP-1 cell line was obtained from the Cell bank of Chinese Academy of Sciences, Shanghai, China. Cells culture reagents including 1640 medium and fetal bovine serum (FBS) were purchased from HyClone (Longan, UT, USA).

After infection with PEDV strains, the obvious cytopathic effect (CPE) of Vero E6 cells was observed at 48 h. Then, the cells were fixed with 4% paraformaldehyde at room temperature for 30 min, followed by blocking with 5% BSA at 37 °C for 1 h. Each hybridoma supernatant was added and incubated overnight at 4 °C. After three washes, the cells were stained with goat anti-mouse IgG H&L (FITC) (1:1000 dilution) (Abcam, UK), followed by incubation for 1 h at 37 °C. Images of the stained cells were then visualized using an inverted fluorescence microscope (DMi8, Leica Microsystems).

BVDV−1 E2 specific monoclonal antibody (mAb) stored in the laboratory. The BVDV FITC conjugate was purchased from VMRD (CJ-F-BVD-10 mL). BVDV−1 mAb E2 gp53 IgG2a Isotype was purchased from VMRD (CATALOG #: 157). BoHV−1 positive serum was stored in the laboratory. Goat Anti-Mouse IgG H&L (FITC) and Goat Anti-Mouse IgG H&L (HRP) were purchased from abcam (ab6785, ab6789). MONTANIDETM ISA 206 VG adjuvant was purchased from Seppic (Shanghai, China). Propolis adjuvant was purchased from Jinhe-Youben (Hohhot, China).