Irdye 700dx conjugated anti rabbit igg

The two ELISA-positive samples selected from late and fast feathering chicken groups were also evaluated with Immunofluorescence assays (IFA) and Western blotting following our previously described method with ALV-J envelope protein specific monoclonal antibody JE9 (kindly provided by Dr. Kun Qian, Yangzhou University) (Venugopal et al., 1997 (link); Dai et al., 2016a (link)). IFA was performed using the FITC-labeled anti-mouse IgG (Sigma, USA) and analyzed by fluorescence microscope using NIS-Elements BR analysis software (Nikon, Japan). IRDye 800-conjugated anti-mouse IgG or IRDye 700DX-conjugated anti-rabbit IgG (1:10,000; Rockland Immunochemicals, USA) were as the secondary antibody in western blot analysis, and analyzed with an Odyssey infrared imaging system (LI-COR Biosciences, USA).

Chicken MDM were infected with a 10⁵ TCID₅₀/mL of ALV-J strain SCAU-HN06. DNA, RNA and total proteins were extracted from the ALV-J infected MDM at 3, 6, 12, 24 and 36 h post-infection (hpi). RT-PCR was employed to detect the ALV-J replication using specific PCR primers H5/H7 [12 (link)]. Western blotting was performed with ALV-J envelope protein specific mouse antibody JE9 (kindly provided by Dr Aijian Qin, Yangzhou University, Yangzhou, China) and rabbit anti-β-actin antibody (Bioworld, Louis Park, USA) according to the method described previously [13 (link)]. IRDye 700DX-conjugated anti-rabbit IgG and IRDye 800-conjugated anti-mouse IgG (Rockland Immunochemicals, Limerick, PA, USA) was used as the secondary antibody. Membranes were visualized and analyzed with an Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA). ALV-J provirus was detected by PCR with primers H5/H7 using DNA template.

Western blotting was performed following our previously described method with ALV-J envelope protein specific mouse anti-monoclonal antibody JE9 (kindly provided by Dr. Aijian Qin, Yangzhou University, Yangzhou, China) and rabbit anti-β-actin antibody [22 (link)]. IRDye 700DX-conjugated anti-rabbit IgG and IRDye 800-conjugated anti-mouse IgG (Rockland Immunochemicals, Limerick, PA, USA) were used as the secondary antibody. Membranes were visualized and analyzed with an Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA).