4 6 diamidino 2 phenylindole dihydrochloride dapi

BxPC-3, CFPAC-1, and PANC-1 cells were plated onto 8-well chamber slides at a concentration of 2 × 10⁴ cells/0.2 mL/well and incubated for 3 days at 37°C with 5% CO₂. The cells were washed three times with serum-free medium and fixed with 4% formaldehyde in PBS at room temperature (RT) for 10 minutes. The cells were washed with PBS containing 10 mM glycine and blocked with Dako Protein Block for 10 minutes. Then, the cells were treated with 5 μg/mL anti-MSLN mAb overnight at 4°C. The cells were washed with PBS and incubated with FITC-labeled anti-mouse IgG (Life Technologies), Alexa Fluor 594-labeled wheat germ agglutinin (WGA) (Life Technologies), and 4′, 6-diamidino-2-phenylindole dihydrochloride (DAPI) (Dojindo, Kumamoto, Japan) for 30 minutes. Cells were imaged using a confocal laser scanning microscope, LSM510 (ZEISS, Oberkochen, Germany). For staining of cancer xenografts, the cancer cells were inoculated to nude mice, as described in the Animal Model Section, and the resulting tumors were taken from the mice, soaked in OCT compound, and frozen. The frozen sections were prepared with cryostat and stained with Alexa Fluor 488-labeled 11-25 mAb, Alexa Fluor 594-labeled WGA, and DAPI.

Immunofluorescence staining was performed on PBMCs. Briefly, cells were incubated with Fc receptor blocking agent (Miltenyi Biotec, Bergisch Gladbach, Germany) and stained with monoclonal antibodies for 20 min at 4 °C in a darkened room. The following monoclonal antibodies were used (all from BioLegend, San Diego, CA, USA): FITC-CD3 clone OKT3, PerCP/Cy5.5-CD14 clone HCD14, APC-CD69 clone FN50, PE-CD142 clone NY2. After fixation of stained cells using Fix/Perm buffer (Thermo Fisher Scientific, Waltham, MA, USA), the suspension of fixed cells was immobilized onto glass slides by cytospin. Nuclei were counter stained with 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI) (DOJINDO, Kumamoto, Japan) in water, and whole sections were mounted in ProLong Diamond (Thermo Fisher Scientific, Waltham, MA, USA). Slides were observed with a confocal fluorescence microscope (FV3000, Olympus, Tokyo, Japan).

The maxillae were dissected and fixed with 4% paraformaldehyde for 6 h at 4°C. After decalcification with 10% ethylenediaminetetraacetic acid (EDTA) for 2 weeks at 4°C, the specimens were embedded in optimal cutting temperature compound (Sakura) and then immediately snap-frozen in liquid nitrogen-cooled isopentane. The frozen sections were cut using a cryomicrotome (Microm) at 6-μm thickness in the buccal-lingual direction. The sections were stained with hematoxylin and eosin (HE) or were used for immunofluorescence staining. For immunofluorescence staining, the frozen sections were air-dried for 10 min, washed with Tris-buffered saline (TBS), and pre-incubated with blocking solution (Dako) for 10 min. The sections were incubated with an anti-integrin β4 goat polyclonal antibody (Cat. No. AF3059; 1:100 dilution; R&D Systems) and an anti-laminin 5 rat monoclonal antibody (Cat. No. ab105472; 1:100 dilution; Abcam) for 2 h at room temperature. After washing in TBS, the sections were incubated for 1 h at room temperature with an anti-rabbit IgG antibody conjugated with Alexa 594 or an anti-rat IgG Alexa 594 of donkey origin (1:200 dilution; Molecular Probes). After counterstaining with 4′, 6-diamidino-2-phenylindole dihydrochloride (DAPI; 1:5000 dilution; Dojindo), all specimens were examined and photographed (Nikon A1 Confocal Microscope System).

Cells cultured on coverslips were fixed with methanol for 10 min at −20 °C, blocked with 2% BSA (Wako) in PBS for 20 min, and then incubated with anti-TRPV4 antibody (#ACC-034, Alomone labs). After 90 min, they were incubated with Alexa Fluor 568-conjugated secondary antibody (#A11012, Life Technologies) in dark for 60 min. Following antibody staining, the nuclei were stained with 1 μg/mL 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; Dojindo, Kumamoto, Japan), and mounted using ProLong Diamond (Life Technologies). Fluorescent images were captured using a Nikon C2 confocal microscope (Nikon, Tokyo, Japan). The images were analyzed using NIS-Elements software version 4.00.06 (Nikon).

The cells were cultured in 24-well plates with a 13-mm cover glass inside. After treatment with NG, OP, or HG medium for 48 h, the cells were fixed in ice cold 4% paraformaldehyde for 15 min, permeabilized with 0.3% Triton X-100 for 15 min, and blocked in Blocking One Histo (Nacalai tesque) for 30 min. The primary antibodies were anti-ATX (1:1000, Clone1F8; Abcam); anti-COL1A1 (1:200; Rockland Immunochemicals); anti-α-SMA (1:500; Dako); anti-rhodamine phalloidin (7:1000; Cytoskeleton, Inc., Denver, CO, USA); anti-ZO-1 (1:1000; Proteintech, Rosemont, IL, USA); anti-β-catenin (1:200; Merck Millipore, Temecula, CA, USA); and anti-E-cadherin (1:200; Bioss, Woburn, MA, USA). The secondary antibodies were Alexa Fluor 488 and 594 (1:1000; ThermoFisher Scientific). The nuclei were stained with 4′,6-diamidino-2′-phenylindole dihydrochloride (DAPI; 1 μg/mL; Dojindo, Rockville, MD, USA). The immunostained slides were observed and images were obtained using a BZ-9000 fluorescence microscope (Keyence, Osaka, Japan).

For imaging of fixed cells, the transduced cells were seeded on an eight-well chamber slide (Matsunami Glass Ind., Ltd., Osaka, Japan) and then fixed with neutral buffered formalin or 1% paraformaldehyde and stained with 1 µg/mL 4',6-diamidino-2-phenylindole, dihydrochloride (DAPI; Dojindo). For live-cell imaging, cells were seeded at 1×10⁵ cells/well on eight-well cover glass chambers (IWAKI, Shizuoka, Japan) and labeled with Hoechst33342 for 20 min before treatment. The images were captured using confocal microscopy (FLUOVIEW FV10i; Olympus, Tokyo, Japan).