Goat anti mouse igg fitc

The cells that grew on the slides were fixed, permeabilized, blocked and incubated with MYC (1:100) (Abcam), p-ANXA2 (Tyr23, 1:100) (Santa Cruz) or His-tag (1:100) (Proteintech) at 4 °C overnight. The bound primary antibodies were detected using goat anti-mouse IgG-FITC or goat anti-rabbit IgG H&L (1:100) (Abcam) at 37 °C for 1 h. The fluorescence was detected via confocal microscopy (General Electric Company, Fairfield, CT, USA).

In all, 5 µl of patient serum was incubated with 90 µl of B. pertussis at an OD_600 nm 0.1 in BB for 45 min with shaking (900 rpm) at 37 °C. Samples were then centrifuged at 3060 × g for 5 min and washed with BB twice before resuspension in 90 µl of BB and 10 µl of IgG-depleted human plasma and incubated for 45 min with shaking (900 rpm) at 37 °C. The plate was then centrifuged and washed two times with BB as described above before resuspending with mouse anti-human C1-INH (Fitzgerald, UK) at 1:1000 in BB. Following 20 min incubation at 4 °C, samples were centrifuged and washed twice more with BB before finally resuspending with 200 µl goat anti-mouse IgG-FITC (Abcam, UK) at 1:500 in BB. Samples were incubated for 20 min at 4 °C, centrifuged and washed twice with BB, and the samples were analysed by flow cytometry.

At 4 and 8 weeks after surgery, longitudinal and cross-sections of the regenerated nerves were cut on a paraffin section system. The sections were incubated with mouse anti-β-tubulin III (1:200, Abcam, Cambridge, UK) at 4°C overnight. Next, the primary antibodies reacted with goat anti-mouse IgG FITC (1:200; Abcam, Cambridge, UK) secondary antibodies for 1 h at 37°C. Then the sections were rinsed and observed under a fluorescence microscope (DM6000; Leica, Wetzlar, Germany).

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.

For immunofluorescence analysis, cells were transferred from U-bottom plates to gelatin-coated microscope slides by cytospine (300xg, 10 min) and perfused by PBS containing 4% (w/v) paraformaldehyde for 20 min at room temperature. Fixed cells were washed with PBS and blocked with 10% rabbit normal blocking serum (Santa Cruz Biotechnology, Dallas, TX, USA), supplemented with 0.3% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) for 45 minutes at RT. Next, cells were washed and double-stained for HSP70 (rabbit polyclonal antibody, 1:500, SPA-812, Enzo Life Sciences, Inc., Farmingdale, NY, USA) and AGO2 (mouse monoclonal antibody, 1:500, B-3, Santa Cruz Biotechnology, Dallas, TX, USA) with PBS supplemented with 1.5% of normal blocking rabbit serum and 0.3% Triton X-100, overnight at 4 °C in wet chamber. Subsequently, cells were washed and secondary fluorescent antibody (goat anti-rabbit IgG-TR and goat anti-mouse IgG-FITC; 1:100 both Abcam, Cambridge, UK), supplemented with 1.5% of normal blocking rabbit serum and 0.3% Triton X-100 was added for 1 h at RT. For fluorescent DNA nuclei staining, DAPI was used (1.5 mg/mL UltraCruz Mounteining Medium, Santa Cruz Biotechnology, Dallas, TX, USA). The intracellular colocalization of AGO2 and HSP70 was analyzed by confocal microscopy (Nikon D-Eclipse C1) using EZ-C1 software.

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.