Donkey anti rabbit igg h l alexa fluor 594

Approximately 1.5 h after the rTMS paradigm, the rats were perfused with 0.9% sodium chloride followed by 4% paraformaldehyde. The brain tissues were fixed with 4% paraformaldehyde for 12 h before transferring into a 30% sucrose solution. Coronal sections were cut on a freezing microtome. The sections (30 μm) were washed with PBS three times, followed by 0.3% Triton X-100 incubation for 10 min and 1% BSA for 1 h at room temperature. Samples were then incubated with rabbit anti-c-fos antibody (1:1000, 226003, synaptic system) at 4°C overnight and then with donkey anti-rabbit IgG H&L (Alexa Fluor® 594) (1:1000, Abcam, USA) for 1 h at room temperature. A confocal laser-scanning microscope (Olympus, FV3000) was used to assess the c-fos expression in different groups.

The expression of SDF-1 and its presence and location were evaluated by IF staining using rabbit anti-SDF-1/CXCL12 polyclonal antibody (Bioss, Woburn, MA, USA). Deparaffinized and rehydrated sections were blocked with 5% bovine serum albumin for 60 min at room temperature (25 °C). Sections were incubated with primary antibody (rabbit anti-SDF-1/CXCL12 polyclonal antibody; Bioss) (1:100 dilution) overnight at 4 °C. Secondary antibodies (Donkey Anti-Rabbit IgG H&L Alexa Fluor® 594; Abcam, Cambridge, UK) (1:200 dilution) were used to incubate the sections for 40 min in room temperature. Quenching of autofluorescence (Vector TrueVIEW; Vector Labs, Burlingame, CA, USA) was performed to remove unwanted fluorescence in the tissue sections following the manufacturer’s protocol. Sections were mounted with VECTASHIELD Vibrance Antifade Mounting Medium with DAPI (Vector Labs, Burlingame, CA, USA), as provided in the kit. Fluorescent images were evaluated within 48 h of mounting using a BZ-X710 fluorescence microscope (Keyence, Itasca, IL, USA).

To examine the SARS-CoV-2 RBD and S1 subunit proteins expression, Vero-E6 cells were infected with the recombinant rLS1-HN-RBD, rLS1-S1-F or rLS1 viruses at a multiplicity of infection (MOI) of 0.5. After 48 hours post-infection (hpi), the cells were fixed with 4% paraformaldehyde for 25 minutes (min), and then the monolayer was washed three times with Dulbecco’s phosphate-buffered saline (DPBS) and permeabilized with Triton 0.1% X-100 for 15 min at room temperature (RT). After washing with the cells with DPBS, the monolayer was incubated with the rabbit polyclonal antibody specific to SARS-CoV-2 RBD protein (1:200) (Sino Biological, Beijing, China), and a chicken antiserum specific to Newcastle disease virus (1:200) (Charles River, Norwich, CT, USA) for 1.5 h at RT. Afterwards, the monolayer was incubated with Donkey Anti-Rabbit IgG H&L-Alexa Fluor® 594 (1:250) and Goat Anti- Chicken IgY H&L-Alexa Fluor® 488 (1:1000) (Abcam, Cambridge, MA, USA) for 60 min at RT. Finally, the cells were developed with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min and observed using an ObserverA1 fluorescence microscope (Carl Zeiss, Germany). Digital images were taken at 400× magnification and processed with the AxioCam MRc5 camera (Carl Zeiss, Germany).

The lung tissue sections were dewaxed and rehydrated, then permeabilized with 0.1% Triton X-100 and blocked with 3% bovine serum albumin for 1 h. The sections were incubated with anti-MPO (sc-52707, Santa Cruz, CA, USA) and anti-citH3 (ab5103, Abcam, Cambridge, UK) antibodies overnight at 4 °C, followed by incubation with the fluorescent secondary antibody for 1 h in the dark. Sections were stained with DAPI solution for 5 min. After being washed with PBS three times, the sections were visualized and recorded using a fluorescence microscope (Nikon digital sight DS-FI2, Japan).
Neutrophils derived from peripheral blood were grown on coverslips pre-coated with 0.1% Poly-L-Lysine for 4 h in the lower chamber, then were fixed with 4% PFA for 10 min, permeabilized with 0.2% Triton X-100 for 10 min. After that, they were incubated with primary antibodies: anti-MPO, anti-citH3, and anti-TOM20 (sc-17764, Santa Cruz, CA, USA) overnight at 4 °C, followed by incubation with the corresponding fluorescent secondary antibody (Donkey Anti-Rabbit IgG H&L (Alexa Fluor® 594) (ab150076, Abcam, Cambridge, UK) and Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) (ab150113, Abcam, Cambridge, UK) in the dark. DAPI was used to stain nuclear. Images were captured utilizing a confocal microscope (Leica TCS SP8, Leica, Germany).

The sections were labeled with an IFN-γ polyclonal antibody (ImmunoWay, Suzhou, China. cat# YT2279) at a 1:500 dilution for 1 h at room temperature and visualized with an Opal 520 Fluorophore (Akoya Biosciences, Massachusetts, U.S.A. Opal 3-Plex Manual Detection Kit, REF: NEL810001KT). Antigen retrieval was subsequently performed. The tissue sections were blocked for 1 h in 5% BSA at room temperature and then labeled with the Rb pAb to IL-1 (Abcam, ab2105) at a 1:500 dilution overnight at 4 °C. Finally, the IL-1 protein antigens were visualized via incubation with donkey anti-rabbit IgG H&L (Alexa Fluor^® 594) (Abcam, ab150076) at a 1:500 dilution for one hour. The images were captured via a Leica TCS SP8 laser confocal microscope.