Anti e coli o plus e coli k antibody coupled to fluorescein isothiocyanate fitc

For microscopy fluorescence analysis, Caco-2 cells were cultured in round coverslips placed at the bottom of 12-well plates and incubated for 24 h at 37°C with 5% CO₂. Bacterial inocula used at an MOI of 10 (1 × 10⁶ CFU) were incubated in the presence of AuNP-LomW, AuNP-EscC, or naive sera (10%) for 1 h at 37°C with slight agitation. After incubation in the presence or absence of sera, bacteria were collected in 1 ml of fresh media and used to infect cell culture plates containing 1 × 10⁵ cells. Additionally, evaluation of bacterial viability was performed by using LIVE/DEAD staining (LIVE/DEAD BacLight bacterial viability kit; Invitrogen, USA) in accordance with the manufacturer’s instructions. For cell infection, monolayers were incubated for additional 2 h at 37°C with 5% CO₂. After infection, cells were washed and fixed with 4% paraformaldehyde–PBS. Polymerized actin was detected by staining with tetramethyl rhodamine isothiocyanate-phalloidin (Molecular Probes-Invitrogen, USA). Cell nuclei and bacteria were mounted using Fluoroshield and DNAs detected with DAPI. EHEC (86-24) was detected by immunofluorescence with anti-E. coli O plus E. coli K antibody coupled to fluorescein isothiocyanate (FITC) (Abcam, USA). Images were taken using an Olympus BX51 upright fluorescence microscope and analyzed by the use of Image J software (University of Wisconsin-Medicine).

For fluorescence microscopy analysis, fixed and permeabilized primary intestinal epithelial cells or primary macrophages were used. Polymerized actin was detected by staining with tetramethyl rhodamine isothiocyanate-phalloidin (Molecular Probes-Invitrogen, USA). DNA from cell nuclei and bacteria were mounted using Fluoroshield and detected with 4′,6-diamidino-2-phenylindole (DAPI). EHEC (86-24) was detected by immunofluorescence with anti-E. coli O plus E. coli K antibody coupled to fluorescein isothiocyanate (FITC) (Abcam, USA). Evaluation of bacteria serum recognition was performed by interaction of EHEC O157:H7 strain 86-24 with sera (1:500) from different immune groups (AuNP-LomW, AuNP-EscC, AuNP-LpfA1, or AuNP-LpfA2) during 1 h at 37°C while shaking. After incubation, cells were washed, fixed, and stained with a goat anti-mouse conjugated to Alexa-488 secondary antibody (1:10,000). Bacterial DNA was visualized using DAPI (Molecular Probes, Invitrogen) and mounted using ProLong Gold antifade (Molecular Probes, Invitrogen). Images were taken using an Olympus BX51 upright fluorescence microscope and analyzed by Image J software (37 (link)).

For microscopy immunofluorescence analysis, infection was performed using the C57BL/6 primary epithelial cells described in the previous section. Following a 4 h infection, the cells were then washed 3 times with 1× PBS and fixed with 4% paraformaldehyde-PBS for 20 min at RT. After another 3 washes with 1× PBS, cells were stained for 1 h at RT with a solution containing PBS with tetramethyl rhodamine isothiocyanate-phalloidin (Invitrogen, Carslbad, CA, USA REF# R415) to visualize polymerized actin and DAPI (Sigma, Cream Ridge, NJ, USA REF# MBD0015) to visualize DNA (1:10,000 for both stains). The cells were washed 3 times with 1× PBS, then stained for 3 h at RT with anti-E. coli O plus E. coli K antibody coupled to fluorescein isothiocyanate (FITC) (1:1000) (Abcam, Cambridge, UK REF# AB20856) to visualize C. rodentium. Coverslips were mounted onto microscope slides with ProLong Gold antifade medium (Invitrogen, Carlsbad, CA, USA), visualized in an Olympus BX51 upright fluorescence microscope, and analyzed using the Image J software (Version is 1.46r).