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.