Fitc conjugated goat anti mouse igg antibody

HCECs or RAW 264.7 (500 μL) cells at a concentration of 2×10⁵/mL were seeded onto 24-well plates with poly-L-lysine–coated slips. When cultured to nearly 80% confluence, the cells were stimulated with 30 μL A. fumigatus hyphae (5×10⁶ CFU/mL) for 16 hours. Then, cells were washed three times with PBST, coated with 4% paraformaldehyde (Solarbio) for 10 minutes, and blocked with 1:10 diluted goat serum (Solarbio) for 30 minutes at room temperature after another three washes with PBST. Purified recombinant nanobodies (100 μL, 1 mg/mL) were added and incubated with cells at 4°C overnight. PBS or aspecific nanobodies of equal volume were used as blank and negative controls, respectively. Subsequently, the HCECs were incubated with 100 μL 1:1,000 mouse antishark VNAR antibodies and 100 μL 1:1,000 FITC-conjugated goat antimouse IgG antibodies (Abcam) at 37°C for 1 hour successively. The RAW 264.7 cells were incubated with 100 μL 1:1,000 rabbit anti–His tag antibodies and 100 μL 1:1,000 FITC-conjugated goat antirabbit IgG antibodies (Abcam) at 37°C for 1 hour successively. Then, PBST was used to wash the cells three times. Cell nuclei were stained with 100 μL 1:100 DAPI for 10 minutes and washed three times with PBST. Fluorescent signals were finally detected with a Zeiss Axiovert microscope at 200× magnification.

PAMs were fixed with cold methanol-acetone (1:1) for 10 min at 4°C, washed three times with phosphate buffered saline (PBS), and blocked with 1% BSA-PBS for 30 min at 37°C. Then, the cells were incubated with SDOW17 (1:200, mAb; Rural Technologies) against PRRSV N protein at 37°C for 1 h. After three washes with PBS, the cells were stained with (FITC)-conjugated goat anti-mouse IgG antibody (1:1000, abcam) at 37°C for 1 h. Finally, the nuclei were stained with DAPI (1:1000, Beyotime) for 5 min. Stainings were observed using fluorescence microscopy.