Las x software version 3

Cells cultured on the fibrous substrates were washed three times. A solution of 4% PFA in PBS was used to fix the cells for 15 min. Permeabilization with 0.2 vol.% Triton X-100 in PBS for 10 min was performed, followed by blocking with 1.0% w/v BSA and 0.1 vol.% Triton X-100 in PBS. Primary antibodies, 4 µg/mL of mouse anti-E-cadherin and 5 µg/mL of mouse anti-α-SMA, in blocking buffer were added for 2 h, followed by the addition of secondary antibody goat anti-mouse AF647 at 1:400 for 1 h. Cell nuclei and F-actin staining with 1 µg/mL DAPI and 13.2 µM Alexa Fluor 488^® phalloidin were performed in PBS for 30 min. All steps were performed at room temperature, with three rounds of PBS washing in between. Coverslips were mounted using Kaiser’s glycerol gelatin and stored at 4 °C in the dark. Images were captured using an inverted confocal laser scanning microscopy system (CLSM, Leica, Stellaris 5, Heerbrugg, Switzerland) outfitted with Power HyD S detectors, a Plan-Apochromat 63x/1.4 Oil CS2 objective (Leica, Heerbrugg, Switzerland), and LAS X software version 3.0 (Leica). Image acquisition was performed sequentially with a field of view of 184.70 μm × 184.70 μm and a pixel density of 1024 × 1024. Three different laser excitation wavelengths were used: 405 nm (DAPI), 488 nm (Alexa Fluor 488), and 633 nm (Alexa Fluor 647).

The T-antigen was evaluated in FFPE tissues from pre-malignant and CRC lesions. Briefly, FFPE tissues were prepared as previously described by us [59 (link)]. After deparaffinization, rehydration, and antigen retrieval, they were incubated with FITC-labeled PNA lectin (1:1000; Vector Laboratories, Newark, CA, USA) for 1 h at room temperature (RT). T24 WT cells were used as negative controls, whereas T-antigen-positive T24 GCNT1 KO cells were used as positive controls [60 (link)]. 4′,6′-diamidino-2-phenylindole dihydrochloride (DAPI, 2.3 × 10⁻³ µg/µL; Thermo Scientific, Waltham, MA, USA) was used as nuclear counterstain. All fluorescence images were acquired on a Leica DMI6000 FFW microscope using Las X software (version 3.0) (Leica, Wetzlar, Germany).