Leica sp5

Immunofluorescence assays were performed on Technovit 8100 sections following the protocol described by us (El-Tantawy et al., 2013 (link); Solís et al., 2016 (link)). Several rat monoclonal antibodies to pectins with high (JIM7, LM20) and low (JIM5, LM19) level of methylesterification, and to various AGP epitopes (LM2, LM6) (PlantProbes, Leeds, United Kingdom) were used (Supplementary Table 1). Sections were treated as follows: PBS for 1 min; 5% bovine serum albumin (BSA) for 5 min; incubation with the primary antibody to pectins or AGPs for 1 h; washing in PBS, three times, 1 min each; incubation with the secondary antibody (anti-rat IgG-conjugated to Alexa 488, Thermo Fisher Scientific, Rockford, IL, United States) diluted 1/25 in PBS for 45 min, in darkness; washing in PBS, three times, 1 min each; staining with DAPI (1 mg/ml) for DNA; washing in PBS and distilled water. After that, sections were mounted in Mowiol and observed in a confocal microscope (Leica SP5, Leica Microsystems, Wetzlar, Germany). In order to make appropriate comparison among fluorescence signals, the same settings for sample excitation and capture of emission were kept in the confocal microscope for each antibody in all samples. Controls were performed by omitting the primary antibody.

For live cell imaging measurements, AsPC-1 cells/chamber were seeded on a four-chamber µslide, with 13 mm glass bottom (ibidi GmbH, Germany). After 24 h cells were transfected with pcDNA3-mutp53R175H, pcDNA3-mutp53R273H or the pcDNA3 empty vector (mock) by using Lipofectamine^TM2000 according to the manufacturer’s instructions. Forty-eight hours after transfection cells were incubated for 30 minutes with a staining solution made of MitoSox Red 1:1000 (Life Technologies) and Mitotracker Green 1:5000 (Life Technologies) in medium without FBS. Before the acquisition, the medium was replaced with a special medium without red phenol (DMEM/F12 NoPhenolRED, Life Technologies) to avoid any interference with the fluorescence signal. Cell images were captured using a confocal laser-scanning fluorescence microscope Leica SP5 (Leica Microsystem, Manheim, Germany) at × 63 magnification and processed using Adobe Photoshop and ImageJ softwares (Rasband, W.S., ImageJ, U. S. National Institute of Health, Bethesda, Maryland, USA (http://rsb.info.nih.gov/ij/, 1997–2008).

Human PASMCs were seeded on chamber slides at a density of 6000/cm² and the next day, exposed to normoxia or hypoxia for 48 h. Cells were fixed with 4% PFA for 10 min at room temperature (RT), washed 3 times for 5 min with PBS, and blocked for 1 h with 3% serum bovine albumin (BSA) in PBS supplemented with 1% Triton X-100. Next, cells were incubated overnight at 4 °C with rabbit anti-AK4 primary antibody (Cat# 30038, GeneTex, Irvine, CA, USA), diluted 1:150 in PBS containing 3% BSA and 0.1% Triton X-100. Afterwards, cells were washed 3 times with PBS for 5 min and incubated for 1.5 h at RT with Alexa Fluor Plus 488 secondary antibody (Cat# A32790, Thermo Fisher Scientific, Dreieich, Germany) diluted 1:400 in PBS containing 3% BSA and 0.1% Triton X-100. Cell nuclei were counterstained with 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI). Finally, slides were mounted with a fluorescent mounting medium (Dako). Fluorescent imaging was performed with a confocal microscope (Leica SP5, Leica Microsystems, Wetzlar, Germany).

ISH images were taken by a light microscope (BX61, Olympus) with a CCD camera (ColorView IIIu, Soft Imaging System, Olympus) and processed by Adobe Photoshop CS3. Fluorescence Z-stacks photos were taken by a confocal laser scanning microscope (Leica SP5, Leica Microsystems) and processed by Imaris 7.6.3 (Bitplane, Zurich, Switzerland).

After obtaining the whole-cell configuration, 20–30 min were allowed for intracellular diffusion of the fluorescent dye. Imaging was performed using a two-photon laser scanning microscope (Leica SP5, Leica Microsystems Inc., Mississauga, ON, Canada) with a mode-locked Ti:Sapphire laser (Chameleon Ultra II, Coherent, Missisauge, ON, Canada) operated at 800 nm wavelength, 80 MHz pulse repeat, 140 fs pulse width. A long-range water-immersion objective (40 ×, numerical aperture 0.8) was used. Fluorescence was detected through external photomultiplier tubes, and images were acquired using the Leica LAS software (Leica Microsystems Inc., Mississauga, ON, Canada). Fluorescence signals were collected by scanning a line along the dendrite of interest (total length: ~5–20 μm). Fluorescence transients were measured at 50–200 μm from the soma. The image focus of the line was carefully checked and occasionally adjusted for possible drift.