Alexa flour 488

The mice in each group were sacrificed 7 days post-surgery. The mice under deep anesthesia were transcardially perfused with ice-cold phosphate-buffered saline (PBS) and followed by 4% paraformaldehyde. Then the brains were quickly removed and fixed in 4% formaldehyde solution at 4°C overnight and dehydrated in 15%−30% sucrose until they sank. The samples were cut into 40-μm sections by a cryostat vibratome. For each brain, every sixth section (anterior and posterior of bregma), covering mainly the striatum, was selected for immunofluorescence to measure the infarct volume. In brief, the sections were incubated with primary antibody of rabbit anti-NeuN (Rabbit polyclonal, 26975-1-AP; Proteintech, USA; 1:500) overnight at 4°C. Then the sections were incubated with appropriated secondary antibody of goat anti-rabbit conjugated to Alexa Flour 488 (Abcam, USA; 1:1,000) for 2 h at room temperature. The images were acquired by a Nikon confocal microscope. The brain loss volume was represented by the loss of neuron. Therefore, the percentage of cerebral infarction volume in mice brain was calculated as the following equation: infarction volume % = (sum of infarction size of each slice)/(sum of area of each slice of brain) × 100%, and the loss area of neuronal cells were quantified by the Fiji ImageJ software.

HK‐2 cells were directly seeded on glass coverslips at low density and then stimulated with IL‐1β (20 ng mL⁻¹) as well as IFN‐γ (50 ng mL⁻¹) for 48 h. Subsequently, cells were coincubated with DiD‐labeled Foe‐TEVs for more than 12 h. HK‐2 cells were then fixed for 20 min in 4% paraformaldehyde in PBS as well as permeabilized for 30 min in 0.25% Triton‐X‐100 in PBS containing 5% bovine serum albumin. After that, at 4 °C overnight, primary antibodies against ICAM‐1 (ab282575; 1:500, Abcam) or VCAM‐1 (1:250, Abcam) and samples incubated together and then at 37 °C for 1 h with fluorescence‐labeled secondary antibodies, including Alexa Flour 488 (1:10 000, Abcam) or Cyanine 5 conjugated secondary antibodies (1:250, Abcam) and the nuclei were counterstained with DAPI (Abcam). Images were captured by confocal microscopy (Zeiss LSM 880) and analyzed by ImageJ software (v1.8.0).

Oocytes and one-cell embryos were fixed in 4% paraformaldehyde in PBS for 15 min at room temperature, permeabilized with 0.2% Triton X-100 for 20 min and blocked with 1% BSA in PBS for 1 h. Oocytes were stained as described above incubating with rabbit polyclonal anti-RIC8 antibody for 4 h at room temperature and using secondary antibody at dilution 1:800. Microfilaments were revealed with Alexa Fluor 488 phalloidin (1:125, Invitrogen), applied along with secondary antibodies.
For co-localization experiments, after staining with goat polyclonal RIC8 antibody (1:30; secondary antibody Alexa Flour 488 donkey anti-goat 1:800) and washes in PBS, oocytes were again blocked in 1% BSA-supplemented PBS for 1 h at room temperature, stained with rabbit polyclonal anti-NuMA (1:100, Abcam), rabbit polyclonal anti-LGN (1:100, Abcam) or mouse monoclonal anti-β-tubulin antibody for 4 h at room temperature. After washes in PBS, secondary antibody Alexa Fluor 555 goat anti-rabbit (1:800, Invitrogen) or Alexa Fluor 633 goat anti-mouse (1:800, Invitrogen) was added. After washes in PBS, oocytes were stained with DAPI (Sigma Aldrich) and mounted with Floromount (EMS). For negative controls primary antibodies were omitted and no staining was observed.

Neutralization assays were performed with live ancestral SARS-CoV-2 viruses in a biosafety level 3 laboratory. VeroE6 cells were seeded in 96-well black plates (SPL Life Sciences, Gyeonggi-do, Republic of Korea). Samples were serially diluted in plain medium and 1000 PFU/well viruses were added for incubation at 37 °C for 1 h. Sample–virus mixtures were then added to the seeded VeroE6 cells and incubated for 1 h at 37 °C. Cells were washed with PBS and replenished with culture medium containing 1% FBS. Inoculated cells were further incubated for 6 h, and then were fixed with 4% formalin. The plates were washed, permeabilized with 0.1% NP40, blocked with 2% BSA, and stained with in-house rabbit anti-SARS-CoV-2 nucleoprotein polyclonal antibody and detected by anti-rabbit Alexaflour 488 (Abcam plc, Cambridge, UK). Fluorescent positive cells were detected by a Biotek Cytation 7 Cell Imaging Multi-Mode Reader (Agilent Technologies, Inc., Santa Clara, CA, USA) with data captured by Gen5 Image Prime version 3.11.19. Neutralization titers were calculated and determined in GraphPad Prism 9 by performing 4-parameter logistical fitting on the detected foci. The 50% focus reduction neutralization titer (FRNT50) was determined as the interpolated reciprocal of the dilution having 50% reduction in infected fluorescent loci compared to control wells.

Animal lung tissues were collected and fixed with 10% formalin for 24 h and paraffin-embedded. Three sections from each animal were used for histology analysis. Upon staining, sections were mounted onto slides and dewaxed in xylene. Antigen retrieval was performed by autoclaving slides at 121 °C for 3 min in Vector antigen unmasking solution, citrate-based (Vector Laboratories, Inc., Newark, CA, USA). Sections were then permeabilized with 0.1% Triton X-100, quenched by Sudan Black B, and blocked with 1% BSA. Infected cells were stained by in-house rabbit anti-SARS-CoV-2 nucleoprotein polyclonal antibody and detected by anti-rabbit Alexaflour 488 (Abcam). Cell nuclei were stained with Hoechst 33258 (Thermo). For H&E staining, tissue sections were stained with Gill’s haematoxylin and eosin-Y. Images were acquired using the Olympus BX53 light microscope (EVIDENT, Tokyo, Japan).

The physical association between RECK and LIFR or gp130 was analyzed by two different but complementary methods: IP/IB as previously described [27 (link), 28 (link)] and double immunofluorescence. For IP, equal amounts of whole cell extracts were incubated with anti-RECK antibodies immobilized on agarose beads under slow rotation. After washing in a buffer containing 50 mM Tris–Cl, 150 mM NaCl, and 0.1% Nonidet P-40 three times, the bound proteins were eluted by boiling in SDS sample buffer, run on SDS–PAGE and IB with LIFR or gp130 antibodies. The antibodies against RECK, LIFR, and gp130 are described above. For immunofluorescence, cells seeded at a density of 2,000 cells on a glass slide were fixed with 4% paraformaldehyde and permeabilized with 0.25% Triton-X100 (30 min at 4°C), washed and then incubated with anti-RECK and anti-LIFR or anti-gp130 antibodies (1 hr at RT) followed by secondary antibodies conjugated with Alexa flour 488 or Alexa flour 594 antibodies (Abcam) and nuclear localizing DAPI (4′,6-diamidino-2-phenylindole dihydrochloride, #D1306, ThermoFisherScientific) at RT for 1 hr. Omission of primary antibodies failed to provide specific signals and served as controls. Images were captured by confocal microscopy (Leica Microsystems, Wetzlar, Germany). Red and green signals were colocalized along with DAPI (blue) in merged images.