Frosted microscope slides

All tissue samples were embedded, sectioned at 5 μm, and mounted on frosted microscope slides (Fisher Scientific) for H&E staining. After dewaxing with xylene, the slides were hydrated with gradient ETOH, stained with hematoxylin and eosin Y solutions, dehydrated in absolute alcohol, cleared in xylene, and mounted with cytoseal Xyl. The stained slides were imaged with a high-performance Nikon microscope (Irving, TX, USA).

The PalmSens4 potentiostat was from PalmSens, The Netherlands. The MoistureMeterSC (stratum corneum moisture measurements) was sourced from Delfin Technologies, Finland. Red Dot 3M Electrodes and the A&D Medical UM‐102B Sphygmomanometer were sourced from Medisave, UK. Microtome blades, frosted microscope slides and 4% Paraformaldehyde (PFA) solution in Phosphate Buffered Saline (PBS) were purchased from Fisher Scientific, UK. Phosphate Buffered Saline tablets (pH 7.4), HistoChoice, concentrated HCl and DPX mounting medium were obtained from Sigma‐Aldrich, UK. The Remington G3 Graphite Razor was from Boots, UK. Haematoxylin (modified Mayer's solution), Bluing reagent and Eosin Y solution were from Abcam, UK. The porcine skin was sourced from a local abattoir, UK.

Vero cells (1 × 10⁶ cells/well) were seeded on cover glass (Deckgläser; Thermo Fisher Scientific) in 6-well plates. Cells were infected with DENV-2 (MOI = 0.1) at 37 °C for 1.5 h, and then washed with PBS before drug treatment for 3 days (for the 72 h virus only group) or 6 days (for all other groups). Cells were then PBS-washed, fixed with 4% formaldehyde, permeabilized using 0.1% Triton X-100, and blocked with 3% BSA in PBS. The DENV-positive foci were analyzed by immunofluorescence staining of the cover glass using primary anti-Flavivirus Group Antigen 4G2 antibody (1:1,000; Millipore) and Alexa Fluor 488 anti-mouse IgG (H + L) secondary antibody (1:500; Invitrogen), followed by treatment with DAPI Fluoromount-G (SouthernBiotech; Birmingham, AL, USA) and sealing on the Frosted Microscope Slides (Thermo Fisher Scientific) with nail polish. Visualization was carried out with a ZEISS LSM 700 confocal laser scanning microscope (ZEISS International; Jena, Germany). Three random viral foci in each treatment group were assessed to measure their diameter as well as determine the number of cells per DENV E-positive focus.

Mice were overdosed on isoflurane gas and perfused with room temperature 0.1M phosphate-buffered solution (PBS), pH 7.3, and then with ice-cold 4% paraformaldehyde (PFA) solution in PBS, 10 days after viral injection surgery. After extraction, the brains were post-fixed in 4% PFA solution overnight. 50 μm thickness slices were obtained using the Integraslice 7550 MM vibrating microtome (Campden Instruments, Loughborough, England), and were stored at 4°C in 0.1M PBS. The slices were mounted onto 25 × 75 × 1 mm frosted microscope slides (Thermo-Scientific, Waltham, MA, United States) using 125 uL ProLong Gold antifade reagent (Invitrogen) as the mountant. The slides were imaged using a Nikon fluorescence microscope (Nikon, Minato, Tokyo, Japan) with images obtained using both 4X and 10X magnification objectives.