4 6 diamidino 2 phenylindole (dapi)

About 5×10⁴ cells in 200 µl of α-MEM (Gibco, Life Technologies, Monza, Italy) were plated in the Matrigel-coated upper chambers of the 24-well Transwell invasion assay plate (Corning, NY 14831 USA). Plates were incubated at 37°C for 48 h. Cells in the lower chamber (including those attached to the lower surface of the membrane) were fixed in 4% paraformaldehyde (VWR, Milan Italy), stained with DAPI (Lonza, Basel, Switzerland), and counted with fluorescence microscope (Metamorph – Axiovert microscope).

Cells were fixed in pre-warmed 4% paraformaldehyde/PBS and permeabilized in 0.2% Triton-X-100/PBS for 10 minutes. Cells were stained with anti-E-cadherin antibody (HECD-1) (M106, Takara Bio Inc., Shiga, Japan) and visualised with Alexa Fluor 488. Nuclei were counterstained with DAPI (Lonza, PA-3013). Images were taken using Axiovert system microscope (Carl Zeiss).

Organoids were washed with PBS and fixed for 15 min with 4% PFA/PBS. Fixation was stopped by rinsing with 100 mM Glycine/PBS. Cells were permeabilized with 0.5% Triton/PBS X-100 and unspecific binding was blocked with blocking solution (5% BSA, 0.2% Triton X-100, 0.05% Tween in PBS) for 90 min. Organoids were then incubated with primary antibody (Rabbit Anti-KRT80 1:200, Sigma-Aldrich) for 2 h, washed three times with washing buffer (0.2% Triton X-100, 0.1% BSA, 0.05% Tween in PBS), and incubated with secondary antibody (Goat Anti-Rabbit Alexa Fluor 555 1:200, Invitrogen) for 45 min. Organoids were washed with immunofluorescence buffer for 20 min and PBS for 10 min. Finally, organoids were mounted in Moviol (AppliChem) containing 5 μg/mL of DAPI (Lonza) and visualized using a Zeiss LSM-780 inverted confocal microscope.

Lung sections were treated with a blocking agent before the incubation with a primary antibody against midkine, and the sections were incubated with primary antibody overnight. This was followed by the treatment with Alexa-555 anti-rabbit secondary antibody (Invitrogen, catalog No. 43957 A) and fluorescein isothiocyanate (FITC) conjugated anti-smooth muscle actin antibody (catalog No. F3777) for 1 h at room temperature, which was accompanied by the incubation with 4′, 6-diamidino-2-phenylindole (DAPI (catalog No. 135–1303); Lonza, Bazel Switzerland) before mounting. The slides were observed under an immunofluorescence microscope (DP-70, Olympus)¹⁵ . Fluorescent intensity was quantified and normalized to the background intensity using ImageJ software (version 1.42; National Institutes of Health, Bethesda, MD; https://imagej.nih.gov/ij/).

Immunofluorescence staining was performed as previously described (8 (link), 10 (link)). The cells were incubated with primary antibodies against Vimentin (Abcam, ab92547, rabbit monoclonal, dilution 1/250) and E-cadherin (Santa Cruz, sc-21791, mouse monoclonal, dilution 1/100) for 1.5 h. Slides were then washed thrice in 0.1% Triton/PBS for 5 min and incubated with the secondary antibodies for 1 h (Millipore, AP124F, goat anti-mouse, FITC conjugated, dilution 1/500; ImmunoReagent, Gtx-Rb-003-DRHO, goat anti-rabbit, TRITC conjugated, dilution 1/500). Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI) (Lonza Group Ltd, Basel, Switzerland), diluted in PBS 1X 1:40,000. Images were visualized on an inverted microscope, Olympus IX51, equipped for fluorescence and phase-contrast microscopy (Olympus, Milan, Italy) and were captured at 40× magnification and acquired with Olympus IX2-LWUCD 6A14956 Digital Camera F-View II (Olympus, Milan, Italy).

To investigate the possible occurrence of apoptosis, a terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling (TUNEL) assay [49] (link) was performed using an in situ apoptosis detection kit (Takara, Kyoto, Japan). All samples were digested with a proteinase K solution (10 µg/ml proteinase K in PBS) for 15 min at room temperature to increase their permeability. After being washed twice with PBS for 5 min, these sections were incubated for 1 h with terminal deoxynucleotidyl transferase and fluorescein isothiocyanate-labeled dUTPs in a humidified chamber at 37°C to label the exposed 3′-hydroxyl ends of fragmented nuclear DNA. After terminating the reaction, the sections were again washed twice with PBS for 5 min. Sections were counterstained with 4, 6-Diamidino-2-phenylindole (DAPI; 30 nM solution in PBT) (Lonza, Walkersville, MD, USA) in the dark for 30 min at room temperature. The stained sections were mounted in Vectasheld (Vector Laboratories, Burlingame, CA, USA) and examined using a fluorescent light microscope (BZ-8100 Keyence, Osaka, Japan).