Dm4000b automated upright microscope system

Different groups of Mφ were washed with PBS and fixed in 4% paraformaldehyde for 10 min. After permeabilizing with PBS containing 0.3% Triton X-100 for 10 min and soaking in 10% goat serum (Solarbio, Beijing, China) with PBS at 37°C for 30 min, cells were incubated with the IL10, IL-12b p40, and IL27 Rα antibody in PBS overnight. Subsequently, cells were then washed with PBS, and the secondary antibody solution mixed with PBS was incubated at 37°C for 1 hour in the dark room. After washing the cells three times with PBS, they were covered with DAPI working solution (Roche Science, Mannheim, Germany). Finally, they were examined using a DM4000B automated upright microscope system (Leica, Wetzlar, Germany).

KYRE-150R Cells were grown on covered slips and treated with 20 μM FH535 (purchased from Calbiochem and dissolved in DMSO) for 24 h. After washing three times with PBS, cells were fixed in 4% paraformaldehyde (Solarbio, Beijing, China) for 20 min at room temperature. The fixed cells were permeabilized with PBS containing 0.3% Triton X-100 (Solarbio, Beijing, China) for 20 min. Then, the cells were soaked in 10% normal goat serum (Solarbio, Beijing, China) with PBS for 30 min. The cells were then incubated with various primary antibodies (β-catenin, E-cadherin, Vimentin,1:200 dilutions) overnight at 4°C. After rinsing in PBS, the cells were then incubated with Alexa Flura® 488 conjugated, goat anti-rabbit IgG (H + L) (Thermo Scientific, Waltham, USA) at 1: 1000 dilution for 1 hr at 37°C at a dark condition. The nuclei were visualized by 1 μg/ml DAPI (Beyotime, Shanghai, China) and incubate for 5 min at room temperature. Images were obtained using a DM4000B Automated Upright Microscope System (Leica, Wetzlar, Germany).