Pannoramic midi system

Pathology was performed on mice sacrificed on day 5 post infection. The lung samples were fixed with 4% paraformaldehyde, embedded in paraffin, and cut into 3.5 μm sections. The fixed tissue samples were stained with hematoxylin–eosin (H&E), and the SARS-CoV-2 antigen was detected by indirect immunofluorescence (IFA). The tissue sections were stained with H&E for routine histology. For IFA, the slides were dewaxed and rehydrated, followed by thermally induced antigen repair within 15 min in the microwave with ethylenediamine tetraacetic acid. The slides were washed with PBS and 0.02% Triton X-100 and then sealed with 5% BSA for 1 h at room temperature. Primary antibody (rabbit anti-SARS-CoV-2 N protein polyclonal antibody, made in-house: 1:500) was dropped onto the slides and then washed in PBS. When the slide was nearly dry, the tissue was covered with Cy3-conjugated goat anti-rabbit IgG (ABCAM, AB6939) at a 1:200 dilution. After washing with PBS, the slides were stained with DAPI (Beyotime) at a 1:100 dilution. The image information was collected using a Pannoramic MIDI system (3DHISTECH, Budapest).

Animal necropsies were performed according to a standard protocol. Tissues for histological examination were stored in 10% neutral-buffered formalin for 7 days, paraffin-embedded, sectioned, and stained with hematoxylin and eosin prior to examination by light microscopy. To examine the SARS-CoV-2 antigen, paraffin dehydrated tissue sections were placed in antigen repair buffer for antigen retrieval in a microwave oven. The tissue was blocked with 5% BSA at room temperature for 1 h, followed by incubation with house-made primary antibody at 1:500 (rabbit anti-CoV N protein polyclonal antibody). After being washed by PBS, the slices were slightly dried and covered with Cy3-conjugated goat anti-rabbit IgG (Abcam) at 1:200 dilution. The slides were stained with DAPI (5 µg/mL) after washing by PBS. The image was collected by Pannoramic MIDI system (3DHISTECH, Budapest, Hungary).

Multiplex immunofluorescence (mIF) staining was performed using Opal 7-Color fIHC Kit (PerkinElmer, Waltham, United States) according to protocols which have been described previously (Parra et al., 2017 (link); Parra et al., 2018 (link)). The embedded tumor tissues that underwent deparaffinization and rehydration were heated at 95°C for 20 min using Tris–EDTA buffer or citrate buffer to retrieve antigen. Next, the slides were incubated with primary antibodies overnight at 4°C. Then, the slides were washed three times with 2-methyl-2H-isothiazol-3-one and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 10 min at room temperature. Next, the slides were incubated at room temperature for 10 min with one of the following Alexa Fluor tyramides included in the Opal 7 kit to detect antibody staining. After BOND Wash Solution washing, the slides were counterstained with DAPI for 5 min to visualize nuclei and then mounted with glycerine. The slides were scanned using the Pannoramic MIDI System (3DHISTECH, Budapest, Hungary). The following primary antibodies are used in this study: CD8 (1:50, Servicebio, Wuhan, China), Ly-6G (1:200, Servicebio, Wuhan, China), and F4/80 (1:200, Servicebio, Wuhan, China).

Lung, brain, and spleen tissues of hamsters were fixed in 10% formalin for 7 days. The formalin solution was replaced twice before being transferred out of the ABSL-4 facility. Then, the samples were embedded in paraffin, sequentially sectioned to 4 μm thickness, and stained with H&E prior to examination by light microscopy. To obtain a representative result, different sites of the lung tissue and almost the whole spleen were sampled for histopathology analysis.
Specific anti-NiV immunoreactivity was detected using anti–NiV N protein rabbit primary antibodies (prepared by our laboratory) at 1:3000 dilution at 4°C overnight. The secondary antibody used was biotinylated goat anti–rabbit IgG (SeraCare, 5220-0336) at a 1:200 dilution for 50 minutes. Slides were developed with diaminobenzidine (DAB) chromogen for 15 seconds and counterstained with hematoxylin for 45 seconds. Images were acquired with a Pannoramic MIDI system (3DHISTECH Ltd.).