Anti sars cov 2 n protein antibody

All compounds except for ciclesonide and EIDD-1931 were purchased from MedChemExpress (Monmouth Junction, NJ, USA). ciclesonide and EIDD-1931 were purchased from Cayman Chemical (Ann Arbor, MI, USA). The following are the lot numbers and purities of each compound: aprotinin (lot, 62009; purity of ≥98.0%), bromhexine hydrochloride (lot, 15159; purity of 99.39%), niclosamide (lot, 15718; purity of 98.68%), ciclesonide (lot, 0472344-2; purity of ≥98.0%), remdesivir (lot, 46182; purity of 99.78%), EIDD-2801 (lot, 67548; purity of 99.94%), and EIDD-1931 (lot, 0590872-1; purity of ≥95.0%). Stock solution was dissolved in dimethyl sulfoxide (DMSO) at a 10 mM concentration. Anti‐SARS‐CoV‐2 N protein antibody was purchased from Sino Biological Inc. (Beijing, China). Alexa Fluor 488 goat anti‐rabbit IgG (H + L) secondary antibody and Hoechst 33342 were purchased from Molecular Probes. Paraformaldehyde (PFA) (32% aqueous solution) and normal goat serum were purchased from Electron Microscopy Sciences (Hatfield, PA, USA) and Vector Laboratories, Inc. (Burlingame, CA, USA), respectively.

RNAscope-IF codetection was performed using the RNAscope^® Multiplex Fluorescent v2 Assay combined with immunofluorescence-integrated codetection (ACD). Paraffin-embedded tissue sections were labeled with an anti-SARS-CoV-2 N protein antibody (Sino Biological, China) at a 1:500 dilution overnight at 4 °C. Then, ISH probes, including V-nCoV2019-S (ACD) and V-nCoV2019-S-sense (ACD), were hybridized to RNA, followed by amplification of the signal operation, and the RNAscope^® Multiplex Fluorescent v2 Assay was run to visualize SARS-CoV-2 N protein antigens via donkey anti-rabbit IgG H&L (Alexa Fluor^® 647) (Abcam, ab150075) at a 1:500 dilution. The images were captured via a Leica TCS SP8 laser confocal microscope.

HAE was gifted from Professor Tan Lab^{62 (link)}. HAE cultures were generated in an air-liquid interface for 4–6 weeks to form well-differentiated, polarized cultures that resemble in vivo pseudostratified mucociliary epithelium. Prior to inoculation, the apical surface of well-differentiated HAE cells was washed three times with PBS. The apical surface of cell cultures was inoculated with MASCp36 and its parental isolate WT (BetaCoV/Beijing/IMEBJ05/2020, Nos. GWHACBB01000000) at an MOI of 0.1 at 37 °C for 2 h, and washed three times with PBS to remove the unbounded virus. HAE cells were cultured at an air-liquid interface at 37 °C with 5% CO₂. At 24, 48, and 72 h post inoculation, 300 μL of PBS was applied to the apical surface of cell cultures and collected after an incubation for 10 min at 37 °C. Viral RNA copies were quantified by real-time qPCR and shown as mean ± SD. WT or MASCp36-infected cells as well as uninfected cells were fixed with 4% paraformaldehyde, permeabilized with Triton X100, and blocked with 10% BSA. The cells were then incubated with anti-SARS-CoV-2 N protein antibody (Sinobiological, 40143-R004, 1:1000) and HRP-conjugated secondary antibody, followed by DAPI staining. The percentage of SARS-CoV-2 N protein positive cells were shown as mean ± SD (n = 4).

Paraffin-embedded tissue sections were dewaxed, antigen repair was performed, the tissue slides were permeabilized with 0.1% Triton X-100 for 15 min and then, the tissue sections were blocked for 1 h in 5% BSA at room temperature (RT). And labelled with an anti-SARS-CoV-2 N protein antibody (Sino Biological, China) at 1:500 dilutions overnight at 4°C. Finally, SARS-CoV-2 N protein antigens were visualized by Alexa Fluor 647-conjugated goat anti-rabbit IgG at a 1:500 dilutions for one hour. The images were captured by a Leica TCS SP8 laser confocal microscope.