EA.hy926 cells were seeded in tissue culture treated glass slides (BD Falcon) and grown at 37uC as described above. After cells reached 100% confluence, the monolayer was washed three times with PBS and medium without antibiotics was added. Four compartments of each slide were inoculated with 1610 7 bacteria (MOI = 10) of either L. biflexa serovar Patoc or L. interrogans serovar Canicola. The remaining four wells were left uninfected to serve as negative controls. In some cases, parallel experiments were performed using cells plated on coverslips in 24 well culture dishes, which allowed centrifugation to facilitate bacterialendothelial cell contact. At the end of the incubation (1 h, 3 h and 24 h) the slides were washed three times with PBS and fixed with 3% (wt/vol) paraformaldehyde in PBS at room temperature for 30 min. Cells were permeabilized with 0.1% Triton X-100 in PBS, washed three more times with PBS, and blocked overnight at 4uC with HEPES buffered saline (HBS) and 1% bovine serum albumin (BSA). On the next day the slides were washed again with PBS and incubated with fresh blocking solution for 1h at room temperature. After blocking, the layers were probed with either rabbit anti-L. interrogans (a gift from Dr. Richard Zuerner, USDA, AMES, IA) diluted 1:5000 or anti-L. biflexa antiserum (Biogenesis, Inc., Brentwood, NH) diluted 1:1000, followed by anti-rabbit IgG-TRITC conjugate (1:1000) plus phalloidin-FITC (200 U/mL) to stain filamentous actin. After repeated washing in PBS, chambers were removed from the slides and Prolong Anti-Fade (Invitrogen, Carlsbad, CA) was used to mount coverslips. Two different microscopes at two different institutions were used throughout the course of this work. At institution one, images were captured using a Zeiss Axioplan microscope with a digital charge-coupled device camera (Hamamatsu, Hamamatsu City, Japan) and co-localization of the fluorescent labels was done using Volocity software (Improvision Inc., Lexington, MA). At the second institution a Zeiss Axioimager Z1 with an Axiocam HrC camera and a Nuance Multi-Spectral Imaging System (software CRI Inc, Woburn, MA, v.2.6.0) was used.