Alex fluor 488 conjugated anti mouse antibody

Formalin-fixed, 30% sucrose solution-dehydrated tissue blocks of colorectal tumors with adjacent noncancerous colorectal mucosa were sliced into 40μm-thick frozen sections, as previously described^{31 (link)}. Sections were incubated with a primary mouse monoclonal antibody against human FPR1 (BD Pharmingen) and a rabbit anti-myeloperoxidase (MPO) antibody (Cell Marque, Rocklin, CA) overnight at 4 °C, rinsed with TBS, and further treated with the secondary antibodies, Alex Fluor^®488-conjugated anti-mouse antibody and Alex Fluor^®568-conjugated anti-rabbit antibody (1:500, Invitrogen), respectively for 1 h in the dark at room temperature. Sections were stained for nuclei with 5 μg/mL 4,6-diamidino-2-phenylindole (DAPI, Beyotime Biotechnology) for 10 min, and then mounted on glass slides. Fluorescent images were taken on a laser-scanning confocal fluorescent microscope (Leica TCS SP8, Leica Microsystems, Wetzlar, Germany). The immunofluorescence intensity was quantified using the ImagePro Plus Software (Media Cybernetics, Silver Spring, MD). The results were expressed as means ± SEM based on a minimum of three individual fields.

RD cells were grown in eight-well chambered slides and grown overnight to a 70% confluency. We pre-treated with the peptides indicated for 1 hr followed by infection with HuCoV-OC43 (at MOI of 0.1) for 1 hr. The cells were washed with media without serum and suspended in medium containing 2% FBS with the same amount of peptides and incubated further for 48 hrs. We then fixed cells with 4% paraformaldehyde for 30 min, followed by permeabilization with PBS containing 1% Triton X-100 for 30 min at room temperature. Cells were blocked in 10% normal goat serum in PBS with 0.5% Triton X-100 for 30 min at room temperature, followed by two washing in PBS with 0.2% Tween-20 (wash buffer), for 10 min each. Mouse monoclonal antibody to HuCoV-OC43 nucleoprotein (Sigma, cat no. MAB9013) was added at a dilution of 1:500 in PBS with 0.2% Tween-20 for 2 hrs at room temperature. Cells were washed 4 times with wash buffer. We incubated with Texas red conjugated secondary anti-mouse antibody (for pJAK2 treatment) and DAPI for 30 min. Antibody treatment was followed by four washings and the addition of mounting media (Southern Biotechnology), and covering with a cover slip. When cells were treated with IFNα−C, we used AlexFluor 488 conjugated anti-mouse antibody (Invitrogen) and DAPI for staining. We imaged cells using a Keyence BZ-X700 fluorescence microscope.