Zen confocal software

H9c2 cells (2 × 10⁴ cells/well) were plated in a 6-well plate and treated as previously described. After treatment, cells were fixed in 4% v/v p-formaldehyde, permeabilized with 0.4% v/v Triton X-100, and blocked with 20% v/v goat serum as previously reported [56 (link)]. Next, cells were incubated overnight at 4 °C with mouse monoclonal antibody against connexin 43 (1:250) and rabbit polyclonal antibody against VDAC1 (1:100). After that, AlexaFluor anti-mouse 488 and anti-rabbit 555 (1:500) were used for 2 h at room temperature (RT) in the dark. Nuclei were detected thanks to Dapi (1:1000). The coverslips were vertically scanned from the bottom by using a 63× (1.40 NA) Plan-Apochromat oil-immersion objective. Images, shown as a single stack, and scale bars were generated with Zeiss ZEN Confocal Software (version number Zen 3.6; Carl Zeiss MicroImaging GmbH, Jena, Germany).

EVs were labelled with BODIPY® FL N-(2-aminoethyl)maleimide. Briefly, 10 μL of Bodipy dye was added to 13.5 mL of S. dominica HR conditioned medium and mixed for 30 sec. After 5 min incubation at room temperature, EVs were isolated by ultracentrifugation as described before. After 24 and 48 h EV treatment, MIA PaCa-2 cells were harvested and used for confocal analysis performed as reported in a previous work^{70 (link)}. Briefly, cells were fixed in p-formaldehyde (4% v/v in PBS; Lonza; Basel, Switzerland), were permeabilized with Triton X-100 (0.4% v/v in PBS; Lonza; Basel, Switzerland), blocked with goat serum (20% v/v in PBS; Lonza; Basel, Switzerland) and then incubated with anti-annexin A1 antibody (rabbit polyclonal; 1:100; Thermo Fisher Scientific; Waltham, MA, USA), overnight at 4 °C. After two washing steps, cells were incubated with anti-rabbit AlexaFluor 555 (1:500; Thermo Fisher Scientific; Waltham, MA, USA) for 2 h at RT in the dark. To detect the nuclei, 4′,6-diamidino-2-phenylindole (DAPI, 1:1000) was used. Samples were vertically scanned from the bottom of the coverslip with a total depth of 5 μm and a 63X (1.40 NA) Plan-Apochromat oil-immersion objective. Images and scale bars were generated with Zeiss ZEN Confocal Software (Carl Zeiss MicroImaging GmbH) and presented as single stack. Confocal microscopy analyses were carried out in three independent experiments.

Reactive oxygen species production in response to mechanical stretch or leptin was detected by dihydroethidium (DHE) staining (10 μM, Sigma-Aldrich). RPV slices (5 μm-thick frozen sections) were incubated with DHE at 37°C, 5% CO₂ for 30 min in a humidified chamber protected from light. Positive DHE intensity signals were quantified using a laser confocal microscope (LSM710, ZEN confocal software Carl Zeiss).

To study the nuclear translocation of NFAT, Rat Aortic Smooth Muscle Cells (RASMCs) were cultured (40 × 10³ per ml) in DMEM media with 10% fetal bovine serum for 3 days and then starved for another day. RASMCs were then treated with leptin (3.1 nM) with or without inhibitors. They were then fixed with freshly prepared 4% paraformaldehyde for 10 min, washed twice with PBS, permeabilized with 0.2% Triton X-100 in PBS with for 20 min, blocked with 1% BSA, 0.1% Triton X-100 in PBS for 10 min, and washed with PBS as described previously (Zeidan et al., 2006 (link)). They were then incubated with NFATc3 antibody (1:100 ratio, Santa Cruz Biotechnology, California, USA) overnight at 4°C followed by secondary antibody Alexa Fluor 594 goat anti-rabbit IgG (1:250 ratio, Santa Cruz Biotechnology, California, USA) for 1 h in darkness at room temperature. To stain F-actin, Phalloidin-Fluorescin isothiocyanate [phalloidin-(FITC); 1 μg/mL, Acti-stain 555 phalloidin, Cytoskeleton, Denver, CO, USA] was added for 20 min. VSMCs were then mounted on glass slides using Mounting Medium (Santa Cruz Biotechnology, California, USA) which contains the nuclear stain DAPI. NFATc3 nuclear translocation was assessed using a laser confocal microscope (LSM710, ZEN confocal software Carl Zeiss).

Transfected or untransfected cells were seeded in 8-well chambers. Twenty-four hours after transfection, cells were treated with or without 5 μg of N-glycolyl MDP 2 for another 24 hours. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100. Samples were blocked with blocking buffer [5% BSA, 2.52 mg/ml glycine in PBST (PBS + 0.1% Tween 20)] and incubated with the primary antibodies diluted in blocking buffer–rabbit anti-LRRK2 1:500 and mouse anti-FLAG 1:250 (for NOD2)–overnight at 4°C. Cells were washed with PBS for 3 x 5 minutes and incubated with secondary antibodies prepared in a diluted (1:5) blocking buffer [Alexa Fluor 488 goat anti-mouse IgG (H+L) 1:1000 and Alexa Fluor 594 donkey anti-rabbit IgG (H+L) 1:1000] for 1 hour. Cells were washed with PBS for 3 x 5 minutes and nuclei were stained with DAPI. Images were obtained by confocal microscopy. Colocalization between LRRK2 and NOD2 was measured from 25–30 cells by Zeiss 2012 ZEN confocal software.

For immunofluorescence, hCLDN3/TOV-112D, TOV-112D, OVCAR-3, and Caov-3 cells were seeded on a 4 well cell culture slide, grown to 80% confluency and treated with 5 μg/mL of human IgG (Jackson Immunoresearch Laboratories) or h4G3 for 1 h at 4 °C. The cell culture slide was washed with PBS, and cells were fixed by 4% formaldehyde for 15 min. Cells were washed with PBS, blocked with PBS containing 5% BSA for 1 h, and incubated with goat anti-human IgG-FITC (Jackson Immunoresearch Laboratories) (1:200 dilution) for 1 h. Cells were washed with PBS, stained with Hoechst 33342 (Invitrogen) for staining nuclei, and mounted using Fluoromount Aqueous Mounting Medium (Sigma–Aldrich, St Louis, MO, USA). Images were taken with the LSM 700 ZEISS laser scanning confocal microscope (Carl Zeiss, Jena, Germany). Data were processed using ZEN confocal software (Carl Zeiss).

Seedlings were grown for 6 d in Petri dishes containing MS medium agar plates supplemented with 1% sucrose and detached cotyledons were analysed. Localization analysis was performed using an inverted laser scanning confocal microscope (LSM780 NLO; Carl Zeiss). For co-localization experiments, detached cotyledons were stained for 15min with 4mM FM4-64 (Life Technologies) (Brandizzi et al., 2004 (link)). The Zeiss ZEN confocal software was used for post-acquisition image processing. An inverted spinning-disc confocal microscope (CarvX, CrEST) was used for the plasmolysis experiments of the GRP-3-RFP seedlings. Imaging was performed using a CFI Planfluo 40× (1.4 Numerical Aperture) oil immersion objective (NIKON) through a 70 µm pinhole disc set at 6 000rpm. Detection was performed using a cooled charge-coupled device CCD camera (CoolSNAP HQ2, Photometrics) and omega band-pass filters XF101-2 (for RFP). The CCD camera, Z-motor, and Confocal head were controlled by Metamorph software (Molecular Devices).