Horseradish peroxidase hrp conjugated goat anti mouse igg antibody

Samples were serial diluted in DMEM–2% FBS and applied onto 90% confluence Vero cell monolayers for 2 h at 37 °C, 5% CO2. After viral adsorption, inocula were removed and replaced by a mixed solution of DMEM–4% FBS and PBS–1% carboxymethyl cellulose in a 1:1 ratio for 3 days. After 3 days, cells were fixated in PBS–4% paraformaldehyde for 15 min at room temperature (RT), and permeabilized with PBS–0.1% Triton for 3 min at RT. Non-specific sites were blocked with PBS–1% bovine serum albumin–0.1% Tween 20 for 30 min at RT. ZIKV envelope protein (E) staining was performed using the mouse anti-E (clone 4G2) antibody as the primary antibody and the horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody (Biorad, Hercules, CA, USA) as the secondary antibody. Finally, freshly prepared peroxidase substrate (Vector Vip; Vector Laboratories) was added for 5–15 min and the foci of infection were manually counted.

The Western blots ere prepared as briefly described. RIPA buffer (50 mM Tris, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, and 1% sodium deoxycholate) containing protease inhibitor and phosphatase inhibitor cocktail (Thermo Scientific) was added to the collected cell pellet, and the resuspended lysates were sonicated as described in the previous paragraphs. The protein concentration was measured using BCA protein assay reagent (Pierce, Rochford, IL). Equal amounts of protein (~20 μg) were separated on 10% SDS-PAGE gels. The proteins were transferred to polyvinylidene difluoride membranes in buffer containing 25 mM Tris, 192 mM glycine and 20% methanol in the cold. The membranes were blocked with 5% bovine serum albumin, incubated with primary antibody overnight at 4 °C and then incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody (1:5,000, 1706516, Bio-Rad) or anti-rabbit IgG (1:10,000, A-11034, Novex, Carlsbad, CA) at room temperature for 1 hour. The visualization was performed with SuperSignal West Pico Chemiluminescent Substrate (34087, Thermo Scientific).

Mouse hybridomas producing the monoclonal antibody 4G2 anti-Flavivirus E protein were purchased from the ATCC (catalog no. HB-112), and a highly-purified antibody preparation was produced by RD Biotech (Besançon, France). Mouse monoclonal anti-JEV NS5 antibody was kindly provided by Dr. Yoshiharu Matsura [29 (link)]. Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody was obtained from Bio-Rad Laboratories (catalog no. 170–6516). Alexa Fluor 488-conjugated goat anti-mouse IgG antibody was obtained from Jackson ImmunoResearch (catalog no. 115-545-003).