Anti mouse il 10 fitc

Mouse anti-E-cadherin, mouse anti-vimentin, mouse anti-STAT3, mouse anti-NF-κB antibodies, and Hoechst were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Cer (from porcine brain) was purchased from Avanti Polar Lipids (Alabama, USA), PA was purchased from Sigma-Aldrich (Darmstadt, Germany), and anti-mouse CD163- PerCP, anti-mouse KI-67-APC, anti-mouse CD68-FITC, and anti-mouse IL-10-FITC were obtained from eBioscience (San Diego, CA, USA). Recombinant mouse IL-4, IFN-δ, and anti-IL-10R inhibitor were purchased from PeproTech (Rocky Hill, NJ, USA).
For immunofluorescence labeling, purified rabbit anti-Vimentin, rabbit anti NF-κB, rabbit anti-E-cadherin, and mouse anti-STAT3, and the secondary antibodies goat anti-mouse and goat anti-rabbit Alexa® Fluor 555 (Thermo Fisher Scientific) were used. Rabbit anti-β-actin (LI-COR Biosciences, Lincoln, Nebraska, USA) were used for immunoblotting.

Lymphocytes were separated from spleen tissue samples using lymphocyte separation medium (Solarbio Biotec, Beijing). The cells were counted, and 5 × 10⁵ to 1 × 10⁶ cells were harvested in cold PBS. The cells were then resuspended in 100 μl PBS and incubated with 0.5 μg anti-mouse CD3a PE, 0.25 μg anti-mouse CD4 FITC, 0.125 μg anti-mouse IFN gamma PE, 0.25 μg anti-mouse IL-4 FITC, 1 μg anti-mouse/rat Foxp3 FITC, 0.25 μg anti-mouse IL-10 FITC or isotype control antibodies (eBioscience, San Diego, CA). The cells were then washed with cold PBS, and 10⁴ cells per sample were analyzed by flow cytometry (CyFlow, PARTEC).