Western Blot Analysis of Tongue Sole Immunity

The head kidney was manually homogenized in radioimmunoprecipitation assay (RIPA) buffer (Sigma, St. Louis, MO, USA) and lysed on ice for 20 min. After centrifugation at 4 °C at 12000 rpm for 10 min, the supernatant was collected, and the protein concentration was quantified with the Enhanced BCA protein assay kit (Beyotime, Shanghai, China). Blood was drawn from the caudal vein of tongue sole, and serum was prepared as described previously [43 (link)]. Head kidney leukocyte protein (50 μg) and serum protein (100 μg) were mixed with SDS-PAGE loading buffer, boiled at 100 °C for 10 min and then separated by SDS-PAGE. The protein bands were transferred from gel onto a polyvinylidene fluoride (PVDF) membrane, and the PVDF membrane was soaked in blocking buffer (5% BSA and 0.05% Tween 20 of PBS, pH 7.2) at room temperature for 1 h. The membrane was then incubated with antibody against rCsCD209 (1:1000 dilution) at room temperature for 1 h followed by extensive washing. The membrane was further incubated with horseradish peroxidase (HRP) conjugated goat anti-mouse IgG (Abcam, Cambridge, Cambridgeshire, UK, 1:5000 dilution) at room temperature for 1 h. After extensive washing, the immune-reactive protein bands were visualized by using an enhanced chemiluminescence kit (Pierce, Rockford, IL, USA).