Sp5 x mp dmi 6000

Colon Chips infected with EHEC-GFP and uninfected controls were washed with PBS and fixed with 4% paraformaldehyde for 2 h. Following fixation, epithelial cells and bacteria were labeled with Alexa Fluor 647 Phalloidin, 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI), and anti-green fluorescent protein-Alexa Fluor 488 conjugate. Images were acquired with an inverted laser-scanning confocal microscope (Leica SP5 X MP DMI-6000) and processed using IMARIS.

For immunofluorescence microscopy, gut chips were washed with PBS, fixed in 4% paraformaldehyde, permeabilized with 0.2% Triton-X (Sigma, USA), blocked with 1% bovine Serum Albumin (Sigma, USA) and incubated for 1 h at 37°C with antibodies against CVB1 (1:100, MAB944; Millipore, USA), Villin (1:100, Abcam, USA) and/or F-Actin (Phalloidin-Alexa-568, Molecular Probes, USA); nuclei were counterstained with DAPI. Fluorescence imaging was carried out using confocal laser scanning microscopy (SP5 X MP DMI-6000, Leica, Germany) and image processing and 3-dimensional Z-stack reconstruction was done using Imaris (Bitplane, Switzerland) and ImageJ (http://imagej.nih.gov/ij/) software. To carry out scanning electron microscopy, gut chips were used in which the top channel was not irreversibly bonded to the membrane, which permitted the device to be dismantled without perturbing the cultured cells. After 7 days of culture, cells were fixed in 2.5% glutaraldehyde (Electron Microscopy, USA), treated with 0.5% osmium tetroxide (Sigma) followed by dehydration in graded ethanol washes. Cells were then dried in a critical point dryer (Tousimis Auto Samdri 815 Series A, Tousimis, Rockville, MD), sputter coated with gold and imaged with a scanning electron microscope (Tescan, Czech Republic).

To carry out immunofluorescence microscopic imaging, the apical and basal channels of the chips were gently washed with PBS using a micropipettor, fixed with 4% paraformaldehyde (Electron Microscopy Sciences) in PBS for 30 min, and then washed with PBS. The fixed samples were permeabilized in PBS containing 0.1% Triton X-100 and 1% Fetal Bovine Serum (FBS) for 30 min at room temperature before filling the channels with staining buffer (1.5% BSA in PBS) containing primary antibodies directed against cleaved caspase-3 (Cell Signaling Technology, 9661 S) or VE-cadherin (Thermo Fisher Scientific, 14–1,449-82), and incubating them overnight at 4°C with gentle shaking. After washing with PBS, the samples were incubated with the corresponding secondary antibodies [Alexa Fluor 647 (Thermo Fisher Scientific, A31573) or Alexa Fluor 555 (Thermo Fisher Scientific, A31570)] for 2 h in the dark at room temperature. Nuclei were stained by adding Hoechst dye (Invitrogen 33,342) to the staining buffer. Fluorescence imaging was performed using a confocal laser-scanning microscope (Leica SP5 X MP DMI-6000) and the images obtained were processed by Imaris software (Bitplane) and analyzed by ImageJ.