Horseradish peroxidase hrp conjugated goat anti mouse antibody

DHAV-1 virulent strain LY0801 (GenBank no. FJ436047) was isolated in 2008 from an outbreak of severe DVH in Shandong province, China (Gao et al., 2012 (link)). BHK-21 cells, HEK 293T cells, and duck embryo fibroblast (DEF) cells were cultured at 37°C in 5% CO₂ in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL of streptomycin sulfate. For liposome-mediated transfection, cells were seeded into 24-well plates and grown in DMEM with 10% FBS and antibiotics to 90% confluence. The anti-DHAV-1 monoclonal antibody (mAb) 4F8 which could recognize the epitope “₇₅GEIILT₈₀” lying in VP1 of DHAV-1 was stored in our lab (Zhang et al., 2015 (link)). Horseradish peroxidase (HRP)- conjugated goat anti-mouse antibody was obtained from Abcam (Cambridge, MA, United States).

Frozen sections of human tonsil, normal, and asthma lung tissue (Biochain) were blocked with 10% goat serum and then stained with anti-SEMA4A mAb (house hold, 1:200 dilution) at room temperature for 2 h. After washing, Horseradish Peroxidase (HRP)-conjugated goat anti-mouse antibody (ab19194, 1:2000 dilution) from Abcam was applied to stain for 15 min. The slides were washed and incubated with substrate DAB Chromogen (D7304, Sigma). Finally, Haematoxylin (H3136, Sigma) was applied to counter stain according to the manufacture’s instruction. For double immunofluorescence staining, the slides were stained with anti-SEMA4A mAb, followed by Alexa Fluor 594-labeled anti-mIgG antibody (A-11020, 1:500 dilution) from Thermo Fisher Scientific. Meanwhile, anti-CD11c Ab (NB110-40766, 1;200 dilution) from Novus Biologicals and Alexa Fluor 488-labeled Goat Anti-Rabbit IgG H&L Abs (ab150077, 1:500 dilution) from Abcam were applied for staining. Images were acquired by using an inverted microscope, BX41 (Olympus, Tokyo).

Cell lysates were subjected to sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE) on a 12% polyacrylamide gel, and the separated proteins were electroblotted onto a polyvinylidene fluoride (PVDF) membrane (Thermo Fisher Scientific). Thereafter, the PVDF membrane was blocked with 5% non-fat milk in Tris-Buffered Saline Tween-20 (TBST) (500 mL NaCl, 0.05% Tween 20, 10 mM Tris-HCl, and pH 7.5) for 1 h. The membrane was incubated at 4 °C for 8 h with anti-DHAV-1 monoclonal antibody (mAb) 4F8 (1:500) [28 (link)], mouse anti-FLuc mAb (Abcam, Cambridge, MA, USA, 1:3000), mouse anti-RLuc mAb (Abcam, 1:3000), mouse anti-Renilla Luciferase mAb (Abcam, ab82968, 1:3000), mouse anti-His-tag mAb (CoWin Bioscience, Beijing, China, 1:3000), and mouse anti-IGF2BP1 mAb (Abcam, 1:3000). The membrane was washed four times with TBST and incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody (Abcam, 1:3000) at 4 °C for 4 h. The PVDF membrane was then visualized with hydrogen peroxide and 3,3′-diaminobenzidine tetrahydrochloride (Sigma-Aldrich, St. Louis, MO, USA).