Axiophot system

Microscopic analyses were conducted using an epifluorescence Axiophot Zeiss system. Images were captured with a cooled CCD camera (Zeiss) and merged using Adobe Photoshop. The images were optimized for best contrast and brightness using the same program, but only those functions that treated all pixels in the image equally.

The bound digoxigenin and biotin probes were detected by incubating the slides in fluoresceinated anti-digoxigenin (Roche Applied Science) and streptavidin-Cy3 (Sigma-Aldrich), respectively, prepared in 5% (w/v) BSA, for 1 h at 37 °C. No immunocytochemical procedures were required for the detection of the Dy-547-labeled telomeric probe. Before staining the DNA with DAPI (4′, 6-diamidino-2-phenylindole), the slides were rinsed for 10 min in 4 × SSC/Tween^{20 (link)} at RT. Finally, the slides were mounted in antifade solution (Vector Laboratories). Hundreds of nuclei were analyzed using an epifluorescence Axiophot Zeiss system. Images were captured with a cooled CCD camera (Zeiss) and merged using Adobe Photoshop. The images were optimized for best contrast and brightness using the same program but only those functions that treated all pixels in the image equally.

Four sets of HeLa cells were cultured on coverslips overnight. Then, the cells were washed with PBS and the medium changed to DMEM containing 0.5% FCS. Afterwards, two sets of cells were intoxicated for 4 h at 37°C, with 20 nM of TcdA1 together with all the indicated BC3-YopT fusion proteins. Then, one set of YopT-treated cells and an untreated set were intoxicated with 4 nM of CNF1 for 1 h. After CNF1 intoxication, the cells were washed twice with PBS, fixed with 4% paraformaldehyde for 15 min, washed again with PBS and permeabilized with 0.15% (vol/vol) Triton X-100 for 10 min. Subsequently, the cells were incubated in the dark with phalloidin–tetramethylrhodamine (TRITC) for 2 h at RT for actin staining. Finally, after washing and treatment with 70% and 100% ethanol, respectively, the cells were dried and embedded with Mowiol supplemented with DABCO (1,4-Diazabicyclo[2.2.2]octane) and DAPI for nuclear staining. Samples were cured for 24 h and the cells visualized using an Axiophot system (Zeiss).