Triton x 100

Renal cancer bulk cells were set down on slides by low speed cytospin. Typically, cells were fixed in 2% paraformaldehyde and permeabilized in 0.1% Triton X-100 (Bio-Rad Laboratories, Richmond, CA /) then incubated overnight at 4 °C with primary antibodies diluted in PBS containing 3% bovine serum albumin (BSA), 0.1% Triton X-100. After two washes in PBS, cells were incubated with Alexa Fluor-conjugated secondary antibodies for 30 min at room temperature in the dark, stained for 15 min with 4,6-diamidino-2-phenylindole (DAPI) (Invitrogen Life Technologies Inc., Grand Island NY), prepared in PBS 3% BSA, and mounted with Prolong-Gold antifade (Invitrogen Life Technologies Inc., Grand Island, NY). Slides were analyzed on a FV1000 confocal-microscope (Olympus, Tokyo, Japan, http://www.olympus-global.com).

Cells of 3–6th passage were attached to L-polylysine-coated glass slides, fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 (Bio-Rad, Hercules, CA, USA) for 20 min at room temperature, and then blocked with 1% bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA) at room temperature for 1 h, and incubated with mouse anti-CD90 (1:100, cat. no. ab134358; Abcam, Cambridge, UK), rabbit anti-alpha smooth muscle actin (1:100, cat. no. A17910; ABclonal, Wuhan, China), mouse anti-fibronectin (1:100, cat. no. ab6328; Abcam, Cambridge, UK) monoclonal antibody conjugated with fluorochrome for 12 h at 4 °C. Then, the cells were incubated for 40 min at room temperature with secondary antibodies (goat anti-mouse IgG conjugated with Alexa Fluor 488, 1:400, cat. no. A11029; Thermo Fisher Scientific, Waltham, MA, USA and goat anti-rabbit IgG conjugated with Alexa Fluor 488, 1:400, cat. no. ab150077; Abcam, Boston, MA, USA) and washed. The cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, Abcam, Cambridge, UK). The actin–cytoskeleton of the cells was stained with phalloidin coupled with Alexa Flour 488 (1:200, cat. no. A12379, Thermo Fisher Scientific, Waltham, MA, USA) for 60 minutes, as per the manufacturer’s instructions. The cells were photographed using an Axio Observer microscope (Carl Zeiss, Oberkochen, Germany).

Cellular CYP3A4 and E-cadherin protein expressions were visually analyzed with immunofluorescence staining and subsequent confocal microscopy. Briefly, on days 1, 7, or 14, cell-laden ICC scaffolds were removed from 24-well plates, washed with PBS, then fixed in 4% paraformaldehyde (PFA) for 5 min. Next, cells were permeabilized with a solution of 0.1% Triton X-100 (Bio-Rad, CA) in PBS for 30 min, washed with PBS, and incubated in 3% BSA blocking buffer for 1 h. Cells were then incubated with mouse monoclonal primary antibodies against CYP3A4 or E-cadherin (Santa Cruz Biotechnology, CA) overnight at 4 °C, washed with PBS, and incubated with anti-mouse secondary antibody conjugated to Alexa Fluor 488 (Life Technologies) for 2 h at room temperature. F-actin was stained with Alexa Fluor 555 phalloidin (Life Technologies). Lastly, cell nuclei were counterstained with 10 μg/ml 4′,6-diamidino-2-phenylindole (DAPI; Life Technologies) for 5 min. Fluorescence images were immediately taken using a confocal microscope.

For the Immunofluorescence (IF) analysis, 10 μm-thick sections of tracheal and bronchial OCT-embedded samples were cut with a cryostat, mounted onto glass slides and fixed with 3% paraformaldehyde (PFA) for 10 min at room temperature (RT) or cold methanol for 10 min at -20 °C. Tracheal and bronchial cells cultured onto glass slides were washed with phosphate buffered saline (PBS 1X) solution and fixed with 3% PFA (10 min at RT) or cold methanol (10 min at -20 °C). Fixed cells were then permeabilized with 0.5%—1% Triton X100 (Biorad) in PBS 1X (10 min at RT), blocked with BSA 2%, incubated with the primary antibodies 30 min at 37 °C (all primary antibodies except for anti-Ki67, incubated overnight at + 4 °C), and thereafter with the corresponding secondary antibodies (30 min at 37 °C). The nuclei were stained with 4',6 diamidino-2-phenylindole (DAPI) and the slide were mounted with Fluorescent Mounting Medium (Dako, Agilent Technologies). Immunofluorescence images were acquired using LSM 900 confocal microscope (Zeiss) and Zen 3.3 software (Zeiss). Cytokeratin 5 (1:1000; Biolegend), Cytokeratin 7 (1:100; Progen), Cytokeratin 14 (1:5000; Biolegend), Ki67 (1:50; Invitrogen), p75NTR (1:50; Cell Signalling), ZO-1 (1:100; Invitrogen), MU5AC (1:50, Progen), and Acetylated $\alpha$ -Tubulin (1:100; Sigma) antibodies were used for immunofluorescence staining.

MIAPaCa2 cells were seeded in 8-well ibi-treat micro-slides (Ibidi, Giemme, Milano, Italy) (30,000/well) in DMEM with 10% FCS. After 48 h, different stimuli were added and incubated for the times indicated. Cells were then fixed with 2% PFA (Sigma-Aldrich) in 4% sucrose for 10 min at room temperature (RT), permeabilized with 0.1% Triton X-100 (Biorad, Segrate, Italy) for 3 min and then incubated for 30 min with 3% BSA (Sigma-Aldrich) at RT. The samples were stained with the primary antibody diluted in 3% BSA overnight at 4 °C followed by incubation with the appropriate secondary antibody for 1 h at RT. Actin was stained with rhodamine phalloidin (Invitrogen, ThermoFisher) at the dilution of 1:40 for 1 h at RT. Nuclei were counterstained with DAPI for 5 min at RT. Images were taken using a Leica SP8 confocal microscope (Leica Microsystems, Wetzlar, Germany).

For immunocytochemistry, 35 µm sections were incubated with a blocking buffer (5% normal serum/ 0.3% Triton X-100, Bio-Rad, Irvine, CA, USA) for 1 h. The tissue was serially sectioned to 35 µm thickness and immunostained with primary antibody [49 (link)]. The sections were incubated with primary anti-Iba-1 (1:200; catalog no. ab87117, abcam), anti-GFAP (1:200; catalog no. MAB360, milipore), and anti-NeuN (1:200; catalog no. 324307s, cell signaling) overnight. Nucleus staining was performed with DAPI. An Axiophot microscope (Leica, TCS SP8, Wetzlar Germany) was used for the analysis of double-stained sections. The immunodensities in the graphs were quantified using the ImageJ program software.