Inverted optical microscope camera system

The xenograft tumors were fixed with 4% formaldehyde, embedded in paraffin, and cut into 4 μm sections. After incubating with EDTA antigen retrieval solution (Beyotime) at 95°C for 15 min, the slides were incubated with endogenous peroxidase blocking solution (Beyotime) at room temperature for 10 min and then were incubated at 4°C overnight with the primary antibodies: anti-E-cadherin, anti-N-cadherin, and anti-vimentin (ab76055, ab76011, and ab92547; Abcam, Cambridge, UK). Thereafter, the slides were incubated with the corresponding secondary antibody (Beyotime) for 30 min at 37°C and orderly stained with diaminobenzidine (Beyotime) and hematoxylin. Finally, the slides were visualized and photographed under an inverted optical microscope-camera system (Olympus).

The 24-well transwell chambers with 8 μm pores (Millipore, Billerica, USA) were used for transwell assays. HeLa cells were seeded into the upper chambers at a density of 1 × 10⁵ and incubated with FBS-free DMEM dissolved with various concentrations of flavonoids in A. conyzoides (50, 200, and 400 μg/mL) or sterile ddH₂O (control). The lower chambers contained the DMEM supplemented with 10% FBS. After incubation for 48 h, cells on the internal surface of the upper chambers were washed with PBS, fixed with 4% paraformaldehyde for 20 min, stained with 0.1% crystal violet (Beyotime, Shanghai, China) for 20 min, and then rinsed with PBS for three times. Finally, three random views for each chamber were captured using an inverted optical microscope-camera system (Olympus), and the number of migration cells in each view was counted. Three independent experiments were performed.