Horseradish peroxidase hrp conjugated goat anti mouse igg

Protein homogenates from goat PBMCs were extracted as previously described (27 (link)). Briefly, the cells were lysed for 20 min on ice in ice-cold lysis buffer (Roche). The lysates were centrifuged at 12,000 × g for 20 min at 4°C to obtain a clear lysate. The protein content of each sample was determined using the BCA Protein Assay Kit (Thermo Scientific). Then, equal amounts of protein were separated on a 12% SDS-polyacrylamide gel and transferred to polyvinylidene difluoride membranes. Membranes were probed overnight at 4°C with an anti-PPRV-N monoclonal antibody provided by China Animal Health and Epidemiology Center (Qingdao, China), or a rabbit polyclonal antibody against sheep SLAM (1:2000; Santa Cruz). The bands were visualized using horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:15000, Boster) or goat anti-rabbit IgG (1:20000, Boster) prior to the ECL protocol (Amersham Biosciences, Piscataway, NJ, USA). As an internal standard, all membranes stripped with primary antibodies were reprobed with anti-β-actin antibody (Invitrogen). Changes in protein expression were determined after normalizing the band intensity of each lane to that of β-actin. Signal was visualized using the Konica SRX 101A developer (Konica Minolta Medical Imaging, Wayne, NJ, USA) and the Quantity One software (Bio-Rad, Mississauga, ON, Canada) was used to densitometrical analysis.

The recombinant proteins were separated by 12% SDS-PAGE and transferred to a PVDF membranes using a Semi-Dry electrophoretic transfer cell (BioRad) for 30 min. The membranes were incubated with 5% (w/v) non-fat for 1 h and then incubated overnight at 4°C with mouse ant-sera against H. taeniaeformis (1:1,000, v/v dilution). Finally, they were washed and incubated with a horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:1,000, v/v dilution; Boster, Wuhan, China) for 1 h. Signals were visualized by exposing X-ray films using Pierce ECL Western Blotting Substrate (ThermoFisher Scientific, USA).

After being grown in 6-well tissue culture plates for 48 h, MARC-145 and pCD163-MARC cells were lysed in ice-cold cell lysis buffer (Beyotime, Shanghai, China) containing 20 mM Tris (pH 7.5), 150 mM NaCl, and 1% Triton X-100 supplemented with phenylmethylsulfonyl fluoride (PMSF; Beyotime, Shanghai, China). The samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Briefly, the samples were resolved in a 12% polyacrylamide gel. Separated proteins were then transferred onto a nitrocellulose membrane and probed with mouse anti-pCD163 MAb or β-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Specific reaction products were detected with horseradish peroxidase- (HRP-) conjugated goat anti-mouse IgG (Boster, Wuhan, China). The membranes were developed using SuperSignal® West Pico Chemiluminescent Substrate according to the manufacturer's suggestions (Pierce, Rockford, IL, USA). Digital signal of chemiluminescent Western blotting was acquired by Bio-Rad GelDoc™ XR & ChemiDoc™ XRS system and analysis was conducted by the Quantity One program, version 4.6 (Bio-Rad). Each experiment was repeated three times.