4 mM in PBS. Plates were incubated overnight at 37°C and substrate degradation was measured by readings at 405 nm. Inhibition of live leptospires binding to laminin or to PLG by recombinant proteins ELISA plates were coated with laminin or PLG (1 μg/well). The plates were washed and blocked with 10% non – fat dry milk in PBS

– T for 2 h at 37°C. The blocking solution was discarded, and the wells were incubated for 90 min at 37°C with increasing concentrations of proteins (0 to 10 μg). After three washings, 50 μL/well of 4 × 107 live low – passage virulent VRT752271 mw L. interrogans serovar Copenhageni strain M20 were added for 90 min at 37°C. The unbound leptospires were washed and the quantification of bound leptospires was performed indirectly by anti – LipL32 antibodies produced in mice (1:4,000), given the fact that LipL32 is a major outer membrane leptospiral protein [28]; the procedure was followed by horseradish peroxidase – conjugated anti – mouse IgG antibodies, essentially as described in Barbosa et al. [6]. The detection was performed by OPD as previously described. Liquid-phase immunofluorescence assay (L – IFA) The localization of LIC11834 and LIC12253 encoded proteins by L – IFA was performed as described by Oliveira et al. [15]. In brief, suspensions of 2.5 ml live leptospires

(~109cells/ml) were harvested at 10,000 rpm for 15 min, washed twice with PBS (with 50 mM NaCl), resuspended in 200 μl of PBS with 6 μg/ml YH25448 supplier propidium iodide Tyrosine-protein kinase BLK to stain the nuclei, and incubated for 45 min at 37°C. After incubation, the leptospires were washed gently with PBS and incubated for 30 min at 4°C with polyclonal mouse anti – serum GSK3326595 price against Lsa33, Lsa25, LipL32 or GroEL at a 1:50 dilution. The leptospires were washed and incubated with goat anti – mouse IgG antibodies conjugated to fluorescein isothiocyante (FITC, Sigma) at a dilution 1:50 for 30 min at 4°C. After incubation with secondary antibody, the leptospires were washed and resuspended in PBS – antifading solution (ProLong Gold, Molecular Probes). The immunofluorescence – labeled leptospires

were examined by employ of a confocal LSM 510 META immunofluorescence microscope (Zeiss, Germany). Nucleotide sequence accession numbers GenBank accession numbers for protein sequences LIC11834 and LIC12235 are AAS70420 and AAS70825, respectively. The protein can also be accessed by the genome nomenclature for the gene locus, LIC number (Leptospira interrogans serovar Copenhageni). ECM and biological components The control proteins fetuin and gelatin, were purchased from Sigma Chemical Co. (St. Louis, Mo.) and Difco®, respectively. Laminin – 1 and collagen Type IV were derived from the basement membrane of Engelbreth – Holm-Swarm mouse sarcoma, cellular fibronectin was derived from human foreskin fibroblasts, plasma fibronectin was isolated from human plasma and collagen Type I was isolated from rat tail.