Donkey anti mouse alexafluor 488 secondary antibody

Cells (10⁵ cells/sample) were fixed with 4% paraformaldehyde for 20 min and permeabilized in 1% v/v Triton X-100 PBS solution. Blocking was performed in 5% v/v Donkey Serum (Sigma-Aldrich) PBS solution for 1h at 37°C. OROV infected and uninfected cells were incubated with mouse polyclonal anti-OROV antibody at 1:300 dilution in blocking solution at 37°C for 30 min. Cells were then washed thrice in PBS and incubated with 2 μg/ml Donkey anti-mouse AlexaFluor 488 secondary antibody (Thermo Fisher Scientific) at 37°C for 30 min. After incubation with the secondary antibody, cells were washed and resuspended in PBS. Flow cytometry was performed in Accuri C6 cytometer (BD Biosciences). At least 10,000 gated events were counted per experimental replica at FITC channel.

In all, 5000 LECs per well were seeded into a 96-well glass-bottom imaging plate (Greiner bio-one). Once confluence was reached, smRNA-FISH was performed using the viewRNA Cell Plus Assay kit (Thermo Fisher Scientific), according to the manufacturer’s instructions. LECs were stained with probes designed to target human LETR1 (Type 1 Probe, VA1-3018146, Thermo Fisher Scientific), human Malat1 (positive control, Type 1 Probe, VA1-11317, Thermo Fisher Scientific), and bacterial DapB (negative control, Type 1 Probe, VF1-11712, Thermo Fisher Scientific). LECs were additionally co-stained with mouse anti-human CD31 antibody (clone JC70A, Dako) at a dilution of 1:50, followed by donkey anti-mouse Alexa-Fluor 488 secondary antibody (Thermo Fisher Scientific) at a dilution of 1:200. Z-stacks of fluorescence images spanning over the entire cell monolayer were acquired using an inverted confocal microscope (Zeiss LSM 780). A self-built macro developed with ImageJ (ver. 2.0.0-rc-69/1.52i)^{108 (link)} was used to quantify the nuclear versus cytoplasmic localization of LETR1 by applying a max intensity projection. After determining the nuclear surface using the Hoechst dye signal, the plugin “analyze particles” was used to count spots present either in the nuclear or cytoplasmic area.