Spinning disc head

DU145 cells were infected with 100 μl of CellLight emGFP-tubulin or 70 μl of TagRFP-tubulin (Life Technologies) in DMEM + 10% FBS lacking phenol red. After 1-2 days at 37 ^oC, cells were imaged using a 100 x Apo TIRF NA 1.49 oil objective, on a Nikon Ti inverted confocal microscope coupled to a Yokogawa Spinning Disc head.

ZIKV-infected monocytes cocultured with either hCMEC/D3 cells or hNPCs grown on coverslips were fixed with 4% PFA, permeabilized with 0.1% Triton X-100 and 0.5% BSA, and labeled with primary antibodies and fluorescent secondary antibodies as necessary. The staining was done in the presence of human FcR-blocking reagent and nuclei were labeled with Dapi. Coverslips were mounted on slides by using Fluoromount Aqueous Mounting (Sigma). Image acquisition was performed on an AxioObserver.Z1 inverted microscope (Zeiss) mounted with a spinning-disc head (Yokogawa), a back-illuminated EMCCD camera (Evolve, Photometrics), and a ×63 or ×100, 1.45 NA oil objective (Zeiss). Images were processed by using FiJi (ImageJ software version 1.51 h) and the 3D reconstruction was performed by using Imaris ×64 version 9.2.

SVG-A cells were grown on glass coverslips in 24-well plates for 24 h, prior to transfection with a plasmid encoding for mNeonGreen-Shh using JetPrime according to the manufacturer’s instructions. 24 h later, cells were washed once with PBS and fixed for 20 min at room temperature (RT) with 4% paraformaldehyde. Blocking and permeabilization was done with PBS-0.1% Triton X-100 and 0.5% BSA for 30 min. Cells were incubated with the indicated primary antibodies for 2 h at RT, followed by incubation with secondary antibodies for 45 min, diluted in blocking buffer (Alexa Fluor Donkey anti-Mouse-647 or Alexa Fluor Donkey anti-Rabbit-647). Coverslips were mounted with Fluoromount (Sigma-Aldrich). Image acquisition was performed using an AxioObserver.Z1 inverted microscope (Zeiss) mounted with a spinning disc head (Yokogawa), a back-illuminated EMCCD camera (Evolve, Photometrics) and a X100, 1.45 NA oil objective (Zeiss) controlled by Visiview v.3.3.0 software (Visitron Systems). For live cell imaging, cells were co-transfected as described above with mNG-SHH and TagRFP-CHMP1A on coverslips in 6-well plates, and imaged 48h later in cell media at 37°C and 5% CO₂ in a dark incubation chamber. Z stacks of 0.5 μm interval with two-channel detection were acquired every 5 s, and time-lapse images were analyzed with Imaris v.9.0.1 (Bitplane).

Vero cells grown on coverslips, infected or non-infected, were fixed with 4% paraformaldehyde and incubated (without permeabilization) with the GB03-A568 antibody specifically recognizing the glycoprotein GPC of JUNV. Then, cells were fixed, permeabilized with 0.5% bovine serum albumin and 0.05% saponin in phosphate-buffered saline, and stained with the mouse monoclonal anti-NP antibody SA02-A647 and 4’,6-diamidino-2-phenylindole (DAPI). The samples were mounted on glass slides and imaged on a Zeiss AxioObserver.Z1 inverted microscope mounted with a spinning disc head (Yokogawa), a QuantEM:512SC EMCCD camera (Photometrics), and a 63 × 1.4 NA oil objective (Zeiss). Each acquisition corresponded to stacks spaced by 0.5 μm that spanned the whole cell volume, and images were analysed using ImageJ (version 1.48d).