Bx53 semi motorized fluorescence microscope

Rehydrated paraffin-embedded lung sections were treated with Antigen Unmasking Solution according the manufacturer’s instruction (Vector Laboratories Inc. Burlingame, CA, USA) to unmask the antigen. After blocking with 1% bovine serum albumin, the sections were incubated with mouse anti-influenza NP, rabbit anti-mouse CD4, or rabbit anti-mouse CD8 primary antibody (all from Abcam) at 4 °C overnight^{39 (link)}, followed by biotin-conjugated secondary antibody (Calbiochem, Darmstadt, Germany) for 30 min at room temperature. Streptavidin peroxidase complex reagent (Vector Laboratories, Burlingame, CA) was then incubated at room temperature for 30 min followed by color development with 3, 3′-diaminobenzidine (DAB, Vector Laboratories). The slides were mounted and examined under microscope. Representative images were captured with Olympus BX53 semi-motorized fluorescence microscope.

IF staining was conducted on deparaffinized and rehydrated lung tissue sections for identification and localization of SARS-CoV-2 nucleocapsid protein (NP), using rabbit anti-SARS-CoV-2-N antibody together with Alexa Fluor 488-conjugated goat anti-rabbit IgG antibody. The lung tissues were treated with antigen unmasking solution in pressure cooker (Vector Laboratories), and blocked with 0.1% Sudan black B for 15 min and 1% bovine serum albumin BSA/PBS at RT for 30 min. This was followed by incubation with primary antibody rabbit anti-SARS-CoV-2-N antibody (Sino Biological 40588-T62, 1:1000 dilution) at 4 °C overnight and Alexa Fluor 488-conjugated goat anti-rabbit IgG antibody (Thermo Fisher Scientific A11034, 1:1000 dilution) for 30 min, and then mounted with 4′,6-diamidino-2-phenylindole (DAPI). The images of the lung tissues were captured with Olympus BX53 semi-motorized fluorescence microscope using cellSens imaging software.

Huh-7 cells were seeded at 4 × 10⁴ cells/well in NuncTM Lab-TekTm 8-well chamber slide (Thermo Fisher Scientific) one day before infection. SFTSV (MOI = 0.05) was pre-incubated with HKU-P1 at the concentration of 1 mg/mL. The slides were fixed at 24 hpi with 10% formalin (BDH, Merck) in phosphate-buffered saline (PBS) for 15 min. The fixed cells were permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) in PBS and blocked with 5% bovine serum albumin (BSA, Sigma-Aldrich) in PBS. The cells were then stained with in-house mouse anti-SFTSV-NP monoclonal antibody (1:2000 dilution with 1% BSA/PBS) and subsequently Alexa488-conjugated goat anti-mice IgG secondary antibody (Thermo Fisher Scientific) (1:2000 dilution with 1% BSA/PBS). The slides were washed thrice with 0.05% Tween-20 (Sigma-Aldrich) in PBS following each antibody incubation steps. The slides were then mounted with coverslips using VECTASHIELD^® Antifade Mountant with 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, California, USA). All slides were examined, and the images were captured with Olympus BX53 semi-motorized fluorescence microscope using cellSens imaging software as previously described [30 (link)].