Fitc conjugated goat anti mouse igg

Vero cells infected with CDV 851 strain, Vero-SLAM cells infected with CDV NJ(11)2 strain, and 293T cells transfected with recombinant H protein were washed once with PBS and fixed in the pre-cooling ethanol absolute for 30 min at 4°C and washed with PBST for three times, followed by incubation in mAbs 2D12, G3N or 2B9 for 1 h at 37°C. After washing in PBST, cells were incubated with the fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (BOSTER, Wuhan, China) for 1 h at 37°C. Finally, the cells were washed and observed under the fluorescence microscopy (Olympus, Japan).

For titration of virus stock, four replicates of 10-fold serially diluted virus (starting from 1/10) were inoculated on MDBK cell monolayer in 96-well culture plates. After 48 h incubation, the culture plates were fixed at 4 °C for 30 min with ice cold absolute ethyl alcohol and subjected to immunofluorescence staining with BVDV-specific mouse monoclonal antibody Mix (RAE2020, AHVLA, UK; 1:200 diluted in PBS) and FITC-conjugated goat anti-mouse IgG (BOSTER, Wuhan, China; 1:200 diluted in PBS). The fluorescence signals were observed undera fluorescence microscopy (ZEISS) and viral titers was expressed as the 50% tissue culture infective dose (TCID₅₀)/mL by Reed-Muench method.

Quadruplicates of 10-fold serially diluted virus samples were added on PK-15 cell monolayer in 96-well culture plates and incubated at 37 °C for 72 h. The plates were then fixed for 30 min with absolute ethyl alcohol at 4 °C and subjected to immunofluorescence staining with the monoclonal antibody (mAb) WH303 (target to E2 protein, AHVLA, UK; 1:200 diluted in PBS) and FITC-conjugated goat anti-mouse IgG (BOSTER, Wuhan, China; 1:200 diluted in PBS). The fluorescence signal was observed under a fluorescence microscopy (ZEISS) and virus titers were calculated by Reed-Muench method and expressed as TCID₅₀ per milliliter.

Vero cells in 24-well cell culture plates were mock infected or infected with PEDV WMB at a multiplicity of infection (MOI) of 0.1 for 24 h. The cells were fixed with 4% paraformaldehyde for 30 min at room temperature (RT), washed three times with PBS, and then permeabilized using 0.5% Triton X-100 for 20 min at RT. Subsequently, the cells were blocked with 10% skimmed milk and then successively incubated with mouse anti-PEDV N mAb (Pulike, Luoyang, China) and FITC-conjugated goat anti-mouse IgG (Boster, Wuhan, China). After incubation at RT for 1 h, the cells were washed with PBS, stained with 4′, 6-diamidino-2-phenylindole (DAPI), and examined with a fluorescence microscope (ZEISS, Germany).

After the pathogenic bacteria (ETEC or S. typhimurium SL1344) or L. fructosus C2 treatment, the medium was removed, and the membranes were excised and fixed in 0.1 M phosphate buffer containing 2.5% glutaraldehyde and 2.0% paraformaldehyde (pH 7.4). The membranes were washed three times in 0.1 M phosphate buffer for 20 min each. The membranes were then fixed in 2% osmium tetroxide and dehydrated in increasing concentrations of acetone. Dehydrated membranes were subsequently infiltrated with epoxy resin, embedded in silicone molds, polymerized and then examined using the transmission electron microscope.
The immunofluorescence staining protocol was adapted from the protocol (Zareie et al., 2005 (link)). Briefly, confluent Caco-2 cell monolayers were rinsed in PBS, and followed by fixation and permeabilization in 5% formaldehyde 20°C for 15 min. Then Caco-2 cells were incubated in 5% (vol/vol) normal goat serum in PBS for 1 h at room temperature and then incubated with primary antibodies against occludin (Zymed, San Francisco, CA) at 37°C overnight. After unbound primary antibodies were rinsed away with PBS, cells or tissues were incubated with secondary FITC conjugated goat anti-mouse IgG (1:500 dilution; Boster) for 1 h at room temperature. Caco-2 cells were thoroughly rinsed with PBS and examined with a fluorescence microscope (Olympus BX5, Japan).

The NA titers of the antibody serums against the parent and mutant HP-PRRSVs on MARC-145 were assayed as described previously [50 (link)] with minor modifications. The viruses were diluted to a concentration of 100 TCID₅₀ per 50 μl (10^3.3 TCID₅₀/ml) in DMEM supplemented with 5 % FBS. Serial dilutions of the neutralizing serums were mixed with each of the viruses and incubated at 37 °C for 1 h. The mixtures (100 μl/well) were transferred to MARC-145 monolayers in 96-well plates and incubated for an additional 2 days at 37 °C with 5 % CO₂. The cells were fixed for 10 min with a solution of 50 % methanol and 50 % acetone. After washing with PBS, expression of the N protein of PRRSV was detected by mAb against the N protein (made in our laboratory) and FITC-conjugated goat anti-mouse IgG (BOSTER, China). The NA titers were determined as the reciprocal of the highest dilution that resulted in more than 90 % reduction of infected cells.

The isolated PAM cells were added to 96-well culture flasks (Costar, Corning Incorporated, NY) and incubated for 6 h at 37 °C in a humidified compartment to allow cells to adhere to flasks. Single replication virus-neutralization test on PAMs was essentially performed as described [19 (link)] with minor modifications. Two-fold serial dilutions of serum N4 in RPMI-1640 were mixed with equal volumes of virus resulting in a final titre of 10⁵ TCID₅₀/ml. RPMI-1640 without serum was included as mock condition. Virus-antibody mixtures were incubated for 1 h at 37 °C and transferred to a 96-well plate (100 μl/well) with PAMs. The inocula were removed after 1 h and replaced by medium, after which the cells were further incubated for another 10 h. The cells were fixed and stained with mAb against the N protein of PRRSV and FITC-conjugated goat anti-mouse IgG (BOSTER, China). The NA titers were determined as the reciprocal of the highest dilution that resulted in more than 90 % reduction of infected cells.