Zen 2 blue edition software

Cells were attached on #1.5 glass coverslips by using Poly-l-lysin, and FISH was performed by using the QuantiGene ViewRNA ISH Assay Kit following the manufacturer’s instruction (Affymetrix) and a specific probe recognizing ZIKV plus strand RNA (Affymetrix VF1-19981). Cell DNA was stained with NucBlue (Thermo Fisher). Images were acquired with an inverted confocal microscope (Zeiss LSM 700) and processed with Zen2 (blue edition) software (Zeiss).

2D cultures and 3D spheroids of ASCs and DFATs were simultaneously stained with the green Calcein AM (Cal-AM, 0.25 µL/mL) dye and red ethidium homodimer-1 (1 µL/mL, EthD-1) dye (Invitrogen). The cells were incubated with a mix of dyes for 30 min in darkness at room temperature and then observed and photographed under fluorescence microscopy Axio Vert.A1 (Carl Zeiss, Oberkochen, Germany). To obtain the precise number of living and dead cells, the 3D spheroids were firstly disrupted with Accutase Cell Detachment (Beckton Dickinson) for 15 min, then stained with Cal-AM + EthD-1 and Hoechst 33342 dye (1 µg/mL, Sigma-Aldrich) for the nuclei staining. Live and dead ASCs and DFATs in both 2D and 3D forms were counted with the ZEN 2 Blue Edition software (Carl Zeiss, Oberkochen, Germany), according to the previously described protocols [11 (link)].

The tissue (1–2 mm²) was fixed in paraformaldehyde 4% (w/v) overnight at 4 °C, wax embedded and stained by H&E following routine procedures. Slides were viewed under an Axiovert 200 inverted microscope linked up to a Zeiss digital 100 camera and images processed using ZEN 2. Blue edition software (Carl Zeiss Microscopy GmbH, Jena, Germany) and Image J.

A549 cells were cultured (3 × 10⁴ cells/well) in an 8-well sterile µ-Slide (Ibidi, Gräfelfing, Germany). The next day, the cells were treated with compound 1 (at both 2 and 20 µM) or with compound 2 (at 20 µM) for 1 h. Afterwards, the cells were irradiated (pre-irradiated) or not (dark conditions) with 700–400 nm light for 1 additional hour. In some experiments, the cells under dark conditions were irradiated for 5 min with the 488 nm fluorescent laser from a confocal microscope. To localize the drug inside the cells, it was excited with 405 nm laser and the emission between 594 and 754 nm was recorded. The differential interference contrast (DIC) image was used to characterize the different cellular structures. Images were taken using a Carl Zeiss LSM 880 spectral confocal laser scanning microscope (Carl Zeiss Microscopy GmbH, Jena, Germany) and analyzed with ZEN 2 blue edition software (Zeiss). Representative images from three independent experiments are shown.

VoC were rinsed with PBS, fixed in PBS, 4% paraformaldehyde for 90 min at room temperature and blocked in PBS, 5% BSA (Sigma-Aldrich) for 1 h at room temperature. VoC were then incubated with the primary antibody in PBS, 0.5% BSA, 0.3% Triton-X100 (Sigma-Aldrich) for 2 h at room temperature, rinsed 3 times for 5 min with PBS and incubated for 1 h at room temperature with the secondary antibody in PBS, 0.5% BSA, 0.3% Triton-X100 and rinsed as above. VoC were post-fixed in PBS, 4% paraformaldehyde for 10 min, rinsed twice for 5 min with PBS and incubated in PBS, 1 µg/mL 4′,6-diamidino-2-phenylindole (DAPI, nuclear dye) for 10 min at room temperature, rinsed twice for 5 min with PBS and stored at 4 °C until observation. Primary antibodies used were anti-vascular endothelial (VE)-cadherin (sc-9989 Santa-Cruz Biotechnologies, Clinisciences, Nanterre, France, 1:250), anti-α-smooth muscle actin-FITC (Sigma-Aldrich F3777, 1:250), anti-zona-occludens 1 (Thermo Fisher Scientific 617300, 1:250). Actin filaments were stained with BODIPY Phalloïdin 558/568 (Thermo Fisher Scientific B-3475, 1:250). Images were taken using a confocal laser scanning microscope (Laser Scanning Microscope 880, LSM 880, Carl Zeiss, Oberkochen, Germany). Images were processed using ZEN 2 blue edition software (Carl Zeiss) and 3D reconstruction images were generated using the 3D viewer plugin from Image J software.