Immunofluorescence imaging
The PET membrane with cells was carefully excised, and washed with PBS twice. Subsequently, the cells were fixed with 4% PFA for 15 minutes. Permeabilization was achieved by treating the cells with 0.2% Triton X-100 for 7 minutes, followed by blocking with goat serum for 40 minutes at room temperature. The samples were then incubated overnight at 4 ℃ with a diluted antibody solution of ZO-1 (Abcam, ab221547, UK), MUC-2 (Abcam, ab11197, UK), Villin (Abcam, ab109516, UK) or Ki67 (Abcam, ab16667, UK), and subsequently with secondary antibodies at room temperature for 1 hour, protected from light. Hoechst (Meilunbio, China) staining was used to visualize the nuclei. For visualization of F-actin, samples were stained with phalloidin (Solarbio, China). Washing with PBS was performed three times. Confocal microscopy (FV-1000; Olympus, Tokyo, Japan) was utilized to capture images. Image processing was conducted using Image J.
H&E staining imaging
The PET membrane with cells was cut off carefully, washed with PBS twice and fixed with 4% PFA for more than 24 hours and dehydrated in a graded series of alcohols in a dehydrator followed by being embedded in paraffin and sliced. Then, the paraffin-embedded sample was dewaxed in an environmentally friendly dewaxing solution, followed by dehydration in a series of alcohol solutions and a final water wash. The sections were stained with Hematoxylin for 3-5 minutes, rinsed with water, differentiated, rinsed again, and counterstained with Eosin for 5 minutes. Then the sections were dehydrated in a series of alcohol solutions and cleared with xylene, and mounted with neutral resin. Finally, the sections were viewed under a microscope, and images were taken for analysis. The cell nuclei appear blue, while the cytoplasm appears red.
SEM imaging
To prepare a sample for SEM imaging, PET membrane with fixed cells were washed with PBS and then fixed with electron microscopy fixative. The sample was then washed with PBS for 3 times (15 min each) and post-fixed with OsO4. After being dehydrated with a series of ethanol concentrations (30%, 50%, 70%, 80%, 90%, 95% ethanol for 15 min, two changes of 100% ethanol for 15 min and isoamyl acetate for 15 min), the sample was dried with a critical point dryer, attached to metallic stubs, and coated with gold before being observed and imaged with a scanning electron microscope.