Prolong anti fade reagent with dapi

RBMVEC were seeded at a cell density of 0.3 × 10⁶ in 35 mm dishes. Tat conjugated VLPs at a final concentration of 10 μg/ml was added to each dish and incubated for 20 min at 37 °C in 5% CO₂. After 3 washes with PBS for 10 min each, cells were fixed using 4% paraformaldehyde (PFA) for 20 min. Coverslips were mounted onto glass slides with ProLong Antifade Reagent with DAPI (Life Technologies). Images were taken using the Perkin Elmer Ultra VIEW ERS spinning disc confocal microscope. Five to ten fields chosen at random were imaged for each sample and representative images are shown for each experiment. DAPI was used to stain for the presence of the nuclei as a counter stain. A Fluorescein isothiocyanate (FITC) filter was employed to image RBMVEC incubated with conjugated VLPs.
To inhibit endocytosis, prior to the incubation with VLPs, cells were incubated with hypertonic sucrose solution (0.45 M) for 15 min at 4 °C and then returned for 120 min at 37 °C and then VLP treatment is given as mentioned above and slides were prepared and imaged.

An In Situ Cell Death Detection kit (TUNEL, Roche) was used to assess whether the viral transduction of MEFs was causing apoptosis. MEFs induced to reprogram were grown on 0.1% gelatin coated 10mm coverslips in 4 well plates were stained according to the manufacturer’s instructions. The stained cells were counterstained using Prolong antifade reagent with DAPI (Life Technologies). Samples were analyzed on a Zeiss LSM 510 Meta confocal microscope.

To determine the seroconversion of animals, all JEV, YFV, and ZIKV IgG indirect immunofluorescence assays (IIFAs) were performed as per the manufacturer’s instructions (Euroimmun, Lübeck, Germany), except for the use of labeled secondary antibody and the mounting agent glycerine, which were replaced by Alexa Fluor 488 goat anti-mouse IgG (A-11029; Thermo Fisher Scientific) and 4′,6-diamidino-2-phenylindole (DAPI; ProLong antifade reagent with DAPI; Thermo Fisher Scientific), respectively. Serum from nonvaccinated animals served as a naive, negative control. Slides were visualized using a fluorescence microscope (FLoid cell imaging station; Thermo Fisher Scientific).

Sodium cholate hydrate (NaC, MW 409 Da) and N-dodecyl-β-D-maltoside (DDM, MW 511 Da) were supplied by Sigma-Aldrich (Copenhagen, Denmark; www.sigmaaldrich.com), Lucifer yellow CH ammonium salt (LY), a fixable analog of the FM lipophilic styryl dye FM 1-43 FX (FM), Texas Red Dextran (MW 3000 Da, lysine fixable) (TRD), ProLong antifade reagent with DAPI, and a monoclonal antibody to Na+/K+-ATPase (α-chain) by Thermo Scientific (Roskilde, Denmark; www.thermodanmark.dk), a rabbit antibody to intestinal alkaline phosphatase (IAP) was from AbD Serotec (Copenhagen, Denmark; www.bio-rad-antibodies.com), and a mouse anti-rat early endosome antigen-1 (EEA-1) antibody by BD Transduction Laboratories (Lyngby, Denmark; www.bdbiosciences.com). A rabbit antibody to pig intestinal aminopeptidase N (ApN) was prepared as previously described [20 (link)].

Frozen slides were permeabilized with 1× TBS + 0.1% Triton-X for 15 minutes at room temperature; they were then washed with 1× TBS. Tissues were blocked with 10% BSA/TBS for 15–20 minutes at room temperature, and slides were incubated with primary antibodies overnight at 4°C. Slides were washed for 10 minutes in TBS at room temperature and incubated with secondary antibodies at room temperature in the dark for 2 hours. Slides were washed twice in 1× TBS and then mounted using ProLong Antifade Reagent with DAPI (Thermo Fisher Scientific, P36931). Supplemental Table 4 includes a list of antibodies used.