4 6 diamidino 2 phenylindole dapi

The paraffin-embedded tissue sections were dewaxing, repairing antigen, and then the tissue sections were blocking for 2 hours in 10% FBS at RT. The slides were then incubated with anti-ACE2 antibody (Abcams, USA) for two hours at 1:500 dilution, RT. After washing three times with PBST, the tissue slides were permeabilized with 0.1% Triton-X100 for 15 min and labeled with antiviral N protein (Sino Biological, China) at 1:500 dilution for two hours at RT. Finally, the ACE2 and SARS-CoV-2 N protein antigens were visualized by Alexa Fluor 647-conjugated goat anti-Rabbit IgG and Alexa Fluor 555-conjugated goat anti-mouse IgG at 1:1000 dilution for one hour, respectively. The cell nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI, Abcam, USA). The images were captured by a Leica TCS SP8 laser confocal microscope.

5-Aminolevulinic acid (ALA) and phorbol-12-myristate-13-acetate (PMA) were purchased from Sigma-Aldrich (St Louis, MO, USA). DCFH-DA was purchased from Beyotime Biotechnology (Beijing, China). GW9662 (1 mM) and N-acetyl-l-cysteine (NAC, 1 mM) from Santa Cruz Biotechnology (Santa Cruz, CA, USA) were added to THP-1-derived macrophages 1 h before sonication. Recombinant human TNF-α protein was purchased from Abcam (Cambridge, UK). For Western blots and immunofluorescence analysis, horseradish peroxidase-conjugated or FITC-conjugated secondary antibodies were purchased from ZSGB-BIO (Beijing, China). 4′6-diamidino-2-phenylindole (DAPI) was purchased from Abcam. All primary antibodies are listed in Supplementary Table 1.

The neurite outgrowth was assessed manually for the SGE on each layer (Figure 2). Sprouted neurites on the tunica surfaces and neurites which grew into the dEAC layers were counted. A neuron-specific staining was performed to evaluate neurite outgrowth by recording confocal laser scanning microscope (CLSM) images with the Leica TCS SP8 stimulated emission depletion (STED) microscope using the confocal imaging mode (objectives: HC PL FLUOTAR 5×/0.15 DRY and HC PL FLUOTAR 10×/0.30 DRY). The neurofilament-specific staining was adapted from Schwieger et al.^{19 (link),22 (link)} The dEAC layers and the SGE were permeabilized with a permeabilization solution (Sigma–Aldrich, St. Louis, MO, USA) containing 0.5% Triton X (Sigma–Aldrich) and TRIS and then blocked using a blocking solution containing 10% serum (FCS; Biochrom) and bovine serum albumin (BSA; Sigma–Aldrich) and 1% Triton. To distinguish SGN from non-neuronal cells, they were stained using a polyclonal antibody against the 200 kD neurofilament (1:1000; Abcam, Cambridge, UK) in combination with the secondary antibody Dylight 488 goat-versus-chicken (Abcam). 4′,6-Diamidino-2-phenylindole (DAPI; Abcam) was used for nucleus labeling.

A549 cells grown in 96-well plates overnight were inoculated with TBEV at a multiplicity of infection (MOI) of 0.1 and different concentrations of ribavirin in fresh culture medium. After 48 h, the medium was removed and the cells were fixed with 4% paraformaldehyde in PBS for 15 min at room temperature, washed twice with PBS, and permeabilized with methanol for 20 min at −20°C. After two washes with PBS and blocking with 3% bovine serum albumin (BSA) in PBS for 2 h, the cells were incubated with formaldehyde-inactivated TBEV immunized mouse ascites recognizing the viral surface antigen (1:500 dilution in 1% BSA) overnight at 4°C. The cells were washed three times with PBS and subsequently labeled with an anti-mouse goat secondary antibody conjugated with Alexa Fluor 488 (1:1,000 dilution in 1% BSA; Abcam, UK) in the dark for 1 h. Finally, the cells were treated with mounting medium with 4^′,6^′-diamidino-2-phenylindole (DAPI) (Abcam, UK) for 5 min to visualize the cell nuclei. The numbers of infected cells and total cells were counted by using Cytation 5 imaging reader (BioTek, United States), and the infection rate was calculated with Gen5 3.10 software. Images were acquired under a fluorescence inverted microscope (Olympus IX81, Japan).

TR146 cells were treated as described above. Cells were fixed with 4% paraformaldehyde followed by permeabilisation with 0.02% Triton-X for 20 min. After washing with PBS, the cells were blocked with 5% bovine serum albumin (Sigma-Aldrich, Missouri, USA) for 1 h at room temperature, followed by incubation with Nrf-2 antibody (Abcam) overnight at 4 °C. Subsequently, the wells were washed and incubated with goat anti-rabbit IgG-Alexa Flour 488 (Abcam) for 1 h and counterstained with 4′6-diamidino-2-phenylindole, DAPI (Abcam) and Phalloidin-iFlour 594 reagent (Abcam) for 5 min each. Stained wells were analysed using a Leica DMi8 fluorescent microscope.