Alexa fluor 647 conjugated goat anti mouse igg h l

In order to study the leukocytes types that could bind to IL-27β, head kidney leukocytes were incubated with 1 μg rOnIL-27β on ice for 30 min. After two times of wash with FACS buffer, mouse anti-His antibody (1:1000, BBI life sciences) and Alexa Fluor 647-conjugated goat anti-mouse IgG H&L (1:2000, Abcam) were sequentially incubated on ice away from light for another 30 min. After incubation, wash with FACS buffer twice. The same method was used for the final staining of the samples with anti-tilapia CD3ε monoclonal antibody. Another control group was not incubated with recombinant protein, but with secondary antibody to eliminate the possibility of non-specific binding. The stained cells were suspended in the flow tube with FACS buffer solution and analyzed by flow cytometry.

The spleen and head kidney leukocytes were stained with mouse anti-tilapia CD28 mAbs (1∶400) in FACS buffer (2% FBS in PBS) on ice for 30 min, then washed twice with FACS buffer. The cells were subsequently incubated with Alexa Fluor 647-conjugated goat anti-mouse IgG H+L (1∶2 000; Abcam, UK) on ice for 30 min, then washed twice. After that, the leukocytes were stained with FITC-conjugated CD3ε mAbs (1∶400) on ice for another 30 min and washed twice. The cells were finally resuspended in FACS buffer and collected on a BD Canto-II flow cytometer. The data were analyzed using FlowJo software v.10.0.7.