Deltavision wide field fluorescence microscope

Vero cells were seeded on cover-slips and treated with 20 μM U18666A for 24 h or with 1 mM MβCD for 4 h. Cells were subsequently infected with RVFV MP-12 at MOI 5 for 16 h and fixed with 4% formalin at room temperature. Cells were blocked with 0.2% Triton X-100 and 2% BSA in PBS at room temperature. Following blocking, cells were incubated with an anti-L antibody (1:500; SinoBiological) for 1 h at room temperature. Cells were washed three times with PBS and incubated with Cy3-conjugated affinipure goat anti-rabbit IgG(H + L) (1:500; Proteintech) for 1 h at room temperature. Cells were washed three times with PBS prior to mounting the coverslips on microscope slides using mounting media containing DAPI (Biotium) for staining of cell nuclei. Images were collected with a DeltaVision wide-field fluorescence microscope (Applied Precision) equipped with a CoolSNAP HQ digital camera (Photometrics) and a 40× objective lens. Excitation light of the required wavelength was generated with an Insight SSI solid-state illumination module (Applied Precision). All acquired images were analyzed using Imaris v. 8.4.1 (Bitplane).

Z-stack images were collected with a DeltaVision wide field fluorescence microscope (Applied Precision, GE) equipped with a digital camera (CoolSNAP HQ; Photometrics) and a 1.4-numerical aperture 100× objective lens. Excitation light was generated with an Insight SSI solid state illumination module (Applied Precision, GE), and the images were deconvolved with SoftWoRx deconvolution software (Applied Precision, GE). In all experiments, identical acquisition conditions were used to acquire all images. Following deconvolution, images were analyzed using Imaris 8.4.1 (Bitplane). An algorithm was designed using the surface function in Imaris to generate surfaces around the signal of interest and the maximum fluorescence intensity detected within these surfaces was measured. The same algorithm was applied to all images within an experiment. For live cell experiments, cells were plated on delta DPG dishes (Thermo Fisher Scientific) and maintained at 5% CO₂ and 37 °C in an environmental chamber on a DeltaVision microscope.