Immunofluorescence Validation of sGLT1 and GLUT2

Immunofluorescence staining was carried out to further validate the effect of FA-g-CS/ANC on the expression of sGLT1 and GLUT2. In short, Caco-2 cells were transfected with NC siRNA (negative control), GLUT2-siRNA-2, and sGLT1-siRNA-3; then, they were seeded onto a 4-well slide and chamber (Watson, Kobe, Japan). Subsequently, cells were incubated with 0.09 µg/mL FA-g-CS, 0.23 µg/mL ANC, or their combination at 37 °C for 48 h. Then, they were fixed with 4% paraformaldehyde for 10 min and soaked with 0.5% Triton X-100 in PBS for 15 min. In this study, goat serum was used to avoid nonspecific binding at room temperature. After 30 min of treatments, the cells were incubated with the primary antibody (β-actin mouse monoclonal (IgG1), Santa Cruz Biotechnology, with 1:100 dilutions) at 4 °C overnight [31 (link)], followed by incubation with the secondary antibody (goat anti-mouse IgG1-HRP, Santa Cruz Biotechnology, Shanghai, China, with 1:100 dilutions) for 1 h at 20–37 °C. Nuclear staining was performed with DAPI, and the images were captured with a fluorescence microscope (BX53, Olympus, Tokyo, Japan).