Tritc labelled phalloidin

Immunofluorescence was performed as previously described [13 (link),22 (link)]. Cells were grown on coverslips in their culture conditions. Once at confluence, cells were treated with protease inhibitor cocktail and Ilomastat. Two hours later TRITC-labelled phalloidin (P1951, Sigma) was applied to the cells to visualize cell morphology and the arrangement of actin cytoskeleton. Nuclei were stained with the fluorescent Hoechst 33342 dye (DAPI) (10μg/ml) (Invitrogen) for 15 min at rt. A single composite image was obtained by superimposition of twenty optical sections for each sample observed. The collected images were analysed by ImageJ software to evaluate co-localization.

Immunofluorescence was performed as previously described [23 (link)]. SFbs were grown on coverslips in their culture conditions. Once at semiconfluence, cells were treated with Th1 (classic and non-classic) stimulated culture supernatants or with TNF-α and IFN-γ. A set of experiments was performed with JIA SFbs. 48h after treatment, leucocytes CFSE-labelled were added to SFbs cells monolayers and incubated in presence or absence of anti-CD106 antibody. After two hours the slides were washed with PBS to eliminate non-adherent cells and were observed on fluorescent microscopy, as described above, to count adherent cells. Then the slides were fixed and TRITC-labelled phalloidin (Sigma, Milano, Italy) was applied to the cells to visualize cell morphology and the leucocyte adhesion on SFbs surface. Nuclei were stained with the DAPI (10μg/ ml) (Invitrogen, Milano, Italy) for 15 min at room temperature. The slides were observed with a Nikon confocal microscopy. A single composite image was obtained by superimposition of twenty optical sections for each sample observed.

MAC-T cells were fixed with 4% paraformaldehyde for 30 min, followed by permeabilization in 0.25% Triton X-100 for 15 min. The cells were then incubated with 50 μM TRITC-labelled phalloidin (Sigma-Aldrich, Saint Louis, MO, USA) in invasion medium for 30 min at room temperature to visualize the actin cytoskeleton. The samples were viewed with a confocal laser scanning microscope (Olympus IX81-FV1000).

The morphology of the cells on the different samples was observed after 1, 7, and 14 days of culture. The control used was TCPS coverslips (Nunc Thermanox Coverslips, ThermoFisher Scientific, Waltham, MA, USA) of 13 mm diameter in a 24-well plate. The number of cells was adjusted according to the well area. The same density of cells was seeded on the hybrids and the TCPS coverslips controls, and after each time point, the cells were fixed with 4% (w/v) para-formaldehyde solution for 15 min and then permeabilized with 0.1% (v/v) Triton X-100 (Sigma Aldrich, St Louis, MO, USA) for 10 min. Nonspecific binding sites were blocked by incubating the disks in Phosphate Buffered Saline (PBS) containing 1% Bovine Serum Albumin (BSA, Sigma Aldrich, St Louis, MO, USA) for 1 h. The cytoskeleton and nuclei of the cells were stained, respectively, with 1:500 diluted TRITC-labelled phalloidin (Sigma Aldrich, St Louis, MO, USA P1951) and 1:1000 diluted 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI, Sigma Aldrich, St Louis, MO, USA D9542) in PBS–BSA 0.5% for 1 h. Each incubation with antibodies was performed in wet and dark conditions. The samples were then washed in PBS–BSA 0.5% and pure water and observed using a LSM710 confocal microscope (Zeiss, Iena, Germany).

After fixation in 4% PFA, organs were stored in 70% ethanol and then rinsed and embedded in paraffin. Next, 4 μm sections were cut and mounted on glass slides. After dewaxing in xylene, graded rehydration in 100%, 95% and 70% ethanol was performed followed by antigen retrieval by boiling in citrate buffer for 20 minutes. Blocking with 10% rabbit or donkey serum as appropriate in 0.1% Triton and with Image enhancer (Invitrogen) was followed by incubation overnight with primary antibodies for VDR (Thermo Fisher), uroplakin III and E. coli at 4°C and Alexa Fluor secondary antibodies (Invitrogen).
For immunocytochemistry, TERT-NHUC were seeded on coverslips (VWR) and grown until nearly confluent. After stimulation with vitamin D and/or E. coli, cells were washed in PBS, then fixed in 4% PFA and processed as described for tissue.
Confocal microscopy was performed using a Leica SP5 system. In order to visualize the actin filament system after infection with E. coli, human uroepithelial cells were stained with tetramethylrhodamine B isothiocyanate (TRITC)-labelled phalloidin (Sigma).