Irdye 800cw conjugated goat anti mouse igg h l antibody

Western blotting was used to detect the PEDV N protein. The treated cells were lysed in NP40 lysis buffer (Beyotime, Shanghai, China) supplemented with Protease Inhibitor Cocktail (Sigma-Aldrich). Equal amounts of the extract were separated by SDS-PAGE, and the proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Merck Millipore, MA, United States). After the membranes were blocked with PBS containing Tween 20 (PBST) and 5% skimmed milk powder, they were incubated with mouse anti-PEDV N mAb (diluted 1:10000) as the primary antibody and then with IRDye®-800CW-conjugated goat anti-mouse IgG (H + L) antibody (diluted 1:10000; Li-Cor Biosciences, Lincoln, NE, USA) as the secondary antibody. The blots were scanned with the Odyssey Infrared Imaging System (Li-Cor Biosciences). The mouse mAb directed against PEDV N was prepared and stored in our laboratory.

CD11b⁺ cells were lysed with lysis buffer (Cytobuster, Novagen, San Diego, CA) for 30 min on ice, The mixture was centrifuged at 12,000 rpm for 30 min at 4°C and protein concentrations were quantified by the BCA protein Assay Kit (Pierce, Rockford, IL, USA). Fifty micrograms of protein of each sample was separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). After blocking with 5% dried milk (wt/vol) in PBS for 1 h at room temperature, each membrane was incubated in the same buffer with CD36 monoclonal Antibody, CD54 (ICAM-1) Monoclonal Antibody (all from ThermoFisher Scientific, Rockford, IL, USA, diluted 1:1,000) or IgG control for overnight at 4°C, this was followed by washing and subsequent incubation with IRDye 800 CW Conjugated goat Anti-mouse IgG (H+L) antibody (Li-COR Biosciences, Lincoln, Nebraska, USA), and detection were carried out using Odyssey (Li-COR).