Mouse anti salmonella lps

For immunofluorescence microscopy, Caco-2 cells were seeded onto glass coverslips and infected as described above. After infection, cells were fixed with 3% paraformaldehyde (PFA) for 15 min at room temperature and washed three times with PBS. Cells were permeabilized for 20 min in 0.1% Triton X-100 and blocked with 5% BSA in PBS for 30 min. Mouse anti-Salmonella LPS (Abcam) was diluted 1:100 in PBS and applied for 1 h. Cells were washed three times with PBS, and then the secondary antibody goat anti-mouse IgG (FITC) (Abcam), diluted 1:200 in PBS, was applied for 1 h. Cells were washed again with PBS and incubated with DAPI (Invitrogen) for 2 min. After washing with PBS, cells were overlaid with 200 μl mounting medium. The cells were inspected for intracellular bacteria using a fluorescence microscope (Olympus) or a confocal laser scanning microscope (Leica) using filter sets for FITC (510 nm excitation, 530 nm emission). Images were further processed using the Leica TCS software package and Adobe Photoshop CS3.

RAW264.7 cells were seeded on glass coverslips and infected as described above. At indicated time points post-infection, the infected cells were fixed with 3% paraformaldehyde (PFA) for 15 min and washed three times with PBS. Cells were then permeabilized for 20 min in 0.1% Triton X-100 solution and washed with PBS, followed by blocking with 5% BSA in PBS for 30 min. The cells were then incubated with mouse anti-Salmonella LPS (Abcam) antibody that was diluted 100 times with PBS for 1 h. After washing the cells three times with PBS, they were incubated with goat anti-mouse IgG (FITC) (Abcam) secondary antibody, diluted 200 times with PBS, for 1 h. After repeating the wash process, the cells were incubated with DAPI (Invitrogen) for 2 min. After a final washing process, cells were covered with mounting medium. A confocal laser scanning microscope (Zeiss LSM800) were used for the observation of intracellular bacteria and image acquisition. The ZEN 2.3 (blue edition) were used for the further image processing.