Cy3 or fitc conjugated goat anti rabbit igg

The transfected cells were seeded in 12-well plates and cultured overnight. Then, the cells were fixed in 4% paraformaldehyde and permeabilized in a solution of 0.1% BSA and 0.5% Triton X-100 at room temperature. After washing to remove the blocking solution, the cells were incubated with primary antibodies against β-catenin, E-cadherin, or vimentin for 60 min at 37°C and then washed twice with 0.1% BSA. After 60 min of incubation at 37°C with Cy3 or FITC-conjugated goat anti-rabbit IgG (Beyotime, Shanghai, China), the cells were counterstained with DAPI and imaged under a fluorescence microscope (Leica).

To detect IL-33 in GC cells and S100A4 in NFs/CAFs, cellular immunofluorescence staining was performed. Briefly, the cells seeded in the culture plates were fastened by 4% paraformaldehyde for 15 min, rinsed in PBS for 15 min, permeabilized with 0.1% Triton X-100 for 10 min, and blocked with 5% goat serum for 1 hour at RT. Next, they were incubated with the primary antibodies, including anti-IL-33 (ab187060, Abcam, 1:200) and anti-S100A4 (ab124805, Abcam, 1:200) for 12 hours at 4°C. Afterward, they were subjected to PBS washing 3 times (5 min each time) and incubated with a Cy3- or FITC-conjugated goat anti-rabbit IgG (Beyotime, Shanghai, China) at RT for 1 hour. The nucleus was stained with DAPI (Beyotime, Shanghai, China) [31 (link)]. A fluorescent microscope (Olympus Corporation; magnification, ×400) was adopted to observe the fluorescence images. Experiments were performed in triplicate.