4 6 diamidino 2 phenylindole dihydrochloride dapi

For immunodetection of ASMA, 1 × 10⁴ cells per cm² were seeded on Nunc Lab-Tek Chambered Coverglass (Thermo Scientific, Waltham, MA, USA) and cultured until 50% confluency. Cells were rinsed with PBS, fixed in 4% paraformaldehyde for 20 min at room temperature (RT), rinsed in PBS, and stored at 4 °C. Cells were permeabilized for 30 min at RT with 0.05% Triton X-100 in PBS complemented with 1% Bovine Serum Albumine (BSA) and 1% Normal Goat Serum (NGS) to reduce non-specific staining. Incubation with an antibody directed against ASMA (MAB1420; R&D System) at a dilution of 1/100 in PBS with 1% BSA and 1% NGS was performed overnight at 4 °C. Cells were washed three times in PBS, followed by incubation with Alexafluor 488 conjugated goat-anti-mouse IgG (Life Technologies, Saint-Aubin, France) at a dilution of 1/500 for 1 h at RT in darkness. After several washes, nuclei were stained with 4′,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI; Life Technologies) at a concentration of 1/40,000 for 10 min at RT in darkness. Images were captured using a Nikon A1RSi confocal laser-scanning microscope (Nis Elements Confocal, Nikon, Amstelveen, The Netherlands).

HEPES (4-(2-hydroxyethyl)–1-piperazineethanesulfonic acid), PEI 25 kDa, heparin sodium salt, Paraformaldehyde solution, FluorSave™ Reagent, Eagle’s Minimum Essential Medium (EMEM), RPMI-1640 Medium, fetal bovine serum (FBS), Penicillin-Streptomycin solution, Dulbecco’s Phosphate Buffered Saline (PBS), trypsin-EDTA solution, 200 mM L-glutamine solution, Paraformaldehyde, Tween20, agarose and Alcian Blue solution (1% in 3% acetic acid pH 2.5) were purchased from Sigma-Aldrich (Darmstadt, Germany). Lipofectamine 2000, SYBR gold dye, AF488-anti-rabbit secondary antibody, rhodamine phalloidin, 4′,6–diamidino–2-phenylindole dihydrochloride (DAPI), Alexa Fluor™ 647 NHS ester and Alexa Fluor™ 488 NHS ester were obtained from Life technologies (Carlsbad, California, USA). Transwell® polyester membrane cell culture inserts (0.4 μm pore size) were purchased from Corning (New York, USA). PneumaCult ALI differentiation medium, hydrocortisone and heparin were purchased from Stemcell Technologies (Vancouver, Canada). Alveofact was purchased from Lyomark Pharma (Oberhaching, Germany). ROTI®GelStain Red and bovine serum albumin were purchased from Carl Roth GmbH (Karlsruhe, Germany). Dicer substrate double-stranded siRNA (DsiRNA) targeting human GAPDH, non-specific DsiRNA and amine-modified siRNA were purchased from integrated DNA Technologies (Leuven, Belgium).

Immunofluorescent staining was performed to detect the distribution of Focal Adhesion Kinase (FAK) and actin filaments (F-actin). Firstly, hGFs were seeded onto 8-well chamber slides (#154534, Lab-Tek^® II Chamber Slide w/Cover, Thermo Fisher Scientific, Rochester, NY) at 5,000 cells/well (7,150 cells/cm²). For the next day, the cells were rinsed with 1.8% NaCl for 2 minutes, 3 times a day. After 24h, the medium was removed and the cells were rinsed 2 times with PBS, fixed in 3.7% formaldehyde in PBS for 20 minutes at room temperature and permeabilized in 0.5% Triton X-100 in PBS for 2 minutes. The cells were then incubated with 2μg/ml of anti-FAK, clone 4.47, Alexa Fluor^® 488 conjugate (#16–233, Merck Millipore, Darmstadt, Germany) in PBS for 1 hour at room temperature. The cells were rinsed with PBS and incubated for 15 minutes with rhodamine-phalloidin (#R415, Invitrogen, Life Technologies Corporation) (1:500 dilution) for F-actin staining. Cells were washed again and counterstained with 4',6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI, #D1306, Life Technologies Corporation) for nuclear staining. After covering the samples with ProLong^® Gold Antifade Reagent (#P36934, Life Technologies Corporation) to protect fluorescent dyes from fading, slides were observed by a fluorescence microscope (Axio Observer.Z1, Carl Zeiss, Oberkochen, Germany).

The paraffin sections of malignant PTs were dewaxed in xylene and rehydrated followed by antigen retrieval in ethylene diamine tetraacetic acid (EDTA) buffer (pH 8.0) by pressure cooking for 2 min. Subsequently, the sections were permeabilized with 0.1% Triton X-100 and incubated overnight at 4°C with rabbit anti-human Snail (Abcam, ab180714, UK), Slug (Abcam, ab180714, UK), Twist (Abcam, ab50581, UK), mouse anti-human GD₂ (Abcam, ab68456, UK) and ALDH1 (Abcam, ab56777, UK) antibodies. Then, the sections were rinsed and incubated with the secondary antibody conjugated to the fluorescent dyes, Alexa Fluor® 488 and Alexa Fluor® 594. Finally, the sections were rinsed and incubated with 4′, 6-diamidino-2-phenylindole dihydrochloride (DAPI; Life Technologies, USA) and visualized under a fluorescence microscope.

Human CRC xenograft tumors (patient-derived xenografts; PDXs) were mechanically and enzymatically dissociated into single-cell suspensions using a Human Tumor Dissociation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany), according to the manufacturer’s instructions. The single-cell suspensions from the PDXs were immunohistologically stained with a mouse anti-human EpCAM antibody (clone EBA-1, Becton, Dickinson and Company (BD), Franklin Lakes, NJ, USA), a mouse anti-human CD44 antibody (clone G44-26, BD). After washing, the cells were resuspended with PBS containing 1% bovine serum albumin, and dead cells were labeled with 1.0 µg/mL 4’,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI; Life Technologies Japan). These samples were analyzed using a FACS Aria II cell sorter (BD).

OCT compound was removed from cryosections by washing for 15 min in phosphate-buffered saline (PBS) at 37°C. Sections were then blocked with 10% (v/v) goat serum, 1% (w/v) bovine serum albumin in PBS containing 0.1% (v/v) Triton X-100 for 1 h at room temperature preceding primary antibody incubation. Primary antibodies were incubated in blocking solution for 2 h at room temperature at the following concentrations: PAX6 (Covance) 1:300, PAX2 (Lifespan Biosciences) 1:100, and Laminin (Abcam) 1:200; Triton X-100 was excluded from the blocking buffer for incubations with anti-Laminin. After several washes with PBS, sections were incubated with Goat anti-rabbit AlexaFluor594 (Life Technologies), 1:800 dilution, in blocking solution at room temperature for one hour, followed by a 1:3000 dilution of the nuclear dye 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI; Life Technologies) for 10 min at room temperature. Slides were mounted following several washes in PBS with Citifluor AF-1 mountant (Electron Microscopy Science).

For immunofluorescence staining, the cultured cells were fixed for 10 min in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 and incubated overnight at 4°C with rabbit anti-human EGFR (#4267s; Cell Signaling Technology) and mouse anti-human CD44 (sc-18849; Santa Cruz Biotechnology, Inc.). Subsequently, the sections were rinsed and incubated with the secondary antibody conjugated to the fluorescent dye Alexa Fluor^® 488 and Alexa Fluor^® 594 (both from Jackson ImmunoResearch, West Grove, PA, USA). The sections were rinsed and incubated with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; Life Technologies, Grand Island, NY, USA). A confocal laser scanning microscope (SP5; Leica, Wetzlar, Germany) was used to visualize the immunofluoresence staining.