Fv1000 fluoview camera

For the histology of the pancreas, paraffin sections of 5 μm were stained with hematoxylin and eosin (Richard Allan Scientific, Kalamazoo, MI). For the immunofluorescence analysis, the frozen sections were incubated for 14 hours at 4 °C with antisera specific for insulin (1/150, guinea pig, Sigma), glucagon (1/2,000, mouse, Sigma), E-cadherin (1/100, rabbit, Abcam, San Francisco, USA), vimentin (1/100, rabbit, Abcam), vimentin (1/100, mouse, Sigma), MafB (1/200, rabbit, Bethyl Laboratories, Montgomery, USA), MafA (1/200, rabbit, Bethyl Laboratories), Pdx1 (1/50, goat, R&D system, Minneapolis, USA), Ngn3 (1/200, rabbit, Millipore), F480 (1/200, rabbit, Santa Cruz, Texas, USA), nestin (1/200, mouse, Abcam) and Ki67 (1/50, mouse, BD, San Diego, CA ). The slides were then incubated for 2 hours at room temperature with species-specific secondary antibodies (1:500; Alexa-594, Alexa-488 or Alexa-405; Invitrogen, Basel, Switzerland). The nuclei were visualized with DAPI (40, 6-diamidino-2-phenylindole) (Sigma). Images were captured with a Fluoview FV1000 camera (Olympus, Tokyo, Japan) and recorded on a computer using the Olympus Fluoview Ver.1.7a viewer.

Immunofluorescence staining was performed to quantify the intracellular leptospires in J774A.1 and THP-1 cells as previously described [30 (link)]. Briefly, coverslips with THP-1 or J774A.1 cell monolayers were infected with L. interrogans strains Lai at an MOI of 100 and were incubated for 4, 12 or 24 h. After three washes with sterile PBS, the cell slips were fixed with 4% paraformaldehyde for 20 min and permeabilized with PBS containing 3% non-fat milk and 0.05% saponin (Sigma). The cell samples were incubated with 1:200 diluted rabbit antiserum against L. interrogans strain Lai for 30 min. After three washes with PBS, the samples were incubated for 30 min with 1:400 diluted Alexa Fluor 568-conjugated goat anti-rabbit F(ab’)2. Finally, the samples were stained with 1 μg ml^-1 DAPI (Invitrogen) for 5 min and examined under a laser scanning confocal microscope (Olympus, Tokyo, Japan). Images were captured with a Fluoview FV1000 camera (Olympus, Tokyo, Japan). Final image processing was performed using the FluoView viewer (version 1.7.a; Olympus). The fluorescence intensity of intracellular leptospires in 100 infected cells was counted. In the confocal microscopic detection, cell monolayers that were pretreated with apocynin (100 μmol/L) as described above were used as a control to determine the potential influence of ROS on the amount of intracellular leptospires.