Rabbit anti sars cov 2 spike

Viruses and control recombinant SARS-CoV-2 spike protein (LakePharma) were denatured with Laemmli sample buffer (Alfa Aesar) by heating at 95 °C for 10 min. Proteins were separated by SDS-PAGE in a 4–15% gradient gel and transferred to polyvinylidene fluoride membranes using a transfer apparatus according to the manufacturer’s protocol (BIO-RAD). After transfer, blots were washed in deionized water and probed using the iBind Flex system according to the manufacturer’s protocol (Invitrogen, Thermo-Fisher). Rabbit anti-SARS-CoV-2 spike (Sino Biological Inc., Beijing, China) was diluted in iBind solution (Invitrogen) at 1:1000. HRP-conjugated anti-rabbit IgG (Jackson ImmunoResearch) was diluted in iBind solution at 1:5000. Blots were washed in deionized water and developed with Azure Biosystems Radiance Plus Femtogram HRP substrate (Azure Biosystems) according to manufacturer’s protocol. The blots were stripped with Restore Western Blot Stripping Buffer (Thermo-Fisher) and reprobed with goat anti-RSV polyclonal antisera (Sigma-Aldrich) and a monoclonal antibody specific for GAPDH (6C5) protein (Thermo-Fisher).

Lung tissues were harvested and used for IF staining [48 (link)]. Briefly, after incubation with blocking buffer (5% normal goat serum) for 10 min at room temperature, the slides were incubated with primary antibodies overnight at 4 °C. After five washes, the sections were incubated with secondary antibody at 37 °C for 1 h. In the in vitro experiment, neutrophils were allowed to attach to the coverslips and incubated for 4 h at 37 °C for immunostaining. The primary antibodies used for IF analysis included rabbit anti-MPO (Abcam, ab9535), rabbit anti-SARS-CoV-2 Spike (Sino Biological, 40589-T62), and recombinant anti-histone H3 (methyl K37) antibodies (Abcam, ab215728). Secondary antibodies included goat anti-rabbit IgG H&L (Alexa Fluor^® 488) (Abcam) and goat anti-rat IgG H&L preadsorbed (Alexa Fluor^® 647) (Abcam, ab150167).

Cells were fixed with 3 % paraformaldehyde for 30 min and subsequently permeabilized with 0.5 % Triton-X for 30 min. After blocking for 1 h with 1 % bovine serum albumin, primary antibody was diluted in 0.5 % bovine serum albumin and the solution incubated overnight at 4 °C. The secondary antibody mixed with DAPI (0.02 mg/ml) was incubated for 1 h at room temperature (RT). Primary antibodies used were as followed: mouse anti SARS-CoV-2 Spike (GeneTex #GTX632604 1:1000), rabbit anti SARS-CoV-2 Spike (Sino Biological #40150-R007 1:1000), rabbit anti IRF-3 (Cell Signaling #4302 1:1000), rabbit anti NF-κB (Cell Signaling #8242 1:1000). Secondary antibodies used were as followed: goat anti mouse Alexa 488 (Invitrogen #A11001 1:1000), goat anti rabbit Alexa 488 (Invitrogen #A11008 1:1000), goat anti rabbit Alexa 647 (Invitrogen, #A21244 1:1000). Images were acquired using the Operetta CLS High Content Analysis System (PerkinElmer) followed by image analysis with Harmony (PerkinElmer).