Goat anti mouse igg conjugated with fitc

E. coli strains including K88ac LT(S63K)ΔSTb, C83902, and DH5α were inoculated in TSB liquid medium and grown for 36 h. One ml of the bacterial solutions was centrifuged at 8,000 g/min for 5 min, washed gently with phosphate buffered saline (PBS) using a wide-bore tip, and resuspended in 1 ml PBS. Five μL of the suspensions was coated on glass slides and immobilized using the heat fixing method. The fixed bacteria were blocked with 5% goat serum for 15 min at room temperature and rinsed with PBS. Then, the slides were incubated with murine monoclonal antibody against a factor of K88 fimbriae diluted in 5% goat serum for 45 min. After being washed with PBS 3 times, the bacteria were incubated with 1:10,000 dilution of goat anti-mouse IgG conjugated with FITC (Abcam, UK) for another 45 min. The glass slides were washed in PBS 3 times, and the dyed bacteria were observed under the oil immersion lens of the microscope.

Cells were seeded in 8-well chamber slides at a density of 1 × 10⁵/ml and cultured for 24 h, and then the medium was replaced with FBS-free medium for a further 24 h. After washing with PBS, the slides were fixed with freshly prepared 4 % paraformaldehyde in PBS for 10 min at room temperature. The slides were permeabilized with 0.1 % Triron X-100 in PBS for 10 min at room temperature. The slides were then blocked for 30 min at room temperature by 1 % skim milk, rinsed, and then incubated overnight with mouse monoclonal anti-MUC5AC (Clone 45 M1) antibody (1:50 dilution; Thermo Fisher Scientific, Fremont, CA) at 4 °C. After washing with PBS, the slides were incubated with goat anti-mouse IgG conjugated with FITC (1:200 dilution; Abcam, Cambridge, UK) for 1 h at room temperature. After washing, the nuclei of the cells were stained with Fluoromount-G containing DAPI (Southern Biotech, Birmingham, AL). The slides were then viewed with a multiphoton confocal LSM 780 NLO microscope system (Carl Zeiss, Jena, Germany) under x400 magnification.