Specific antigen detection by immunofluorescence Detection of 20-kDaPS and PIA by immunofluorescence was performed, as previously described [7, 70]. Briefly, overnight cultures of S. epidermidis strains in TSB were diluted 1:100 in 2 mL fresh medium and incubated for VX-765 mouse 18 h at 37°C with shaking. After brief vortex, bacterial suspensions were adjusted to approximate absorbance578 0.2 (Spectrophotometer, Novaspec Plus) and aliquots (10 μL per well) were applied to immunofluorescence slides (CA Hendley Essex Ltd, Essex, United Kingdom). Slide preparations were air-dried, fixed

with cold acetone and stored at 4°C until use. Aliquots (20 μL per field) PIA or 20-kDaPS antisera diluted 1:50 in PBS were applied

to slides which were incubated for 30 min at 37°C. After washing three times with PBS, 10 μL of fluorescein-conjugated anti-rabbit immunoglobulin G (Sigma, UK) diluted 1:80 in phosphate buffered saline were applied, and slides were incubated for 30 min at 37°C. After washing, they were mounted using Vectashield and viewed with a Zeiss AxioImager fluorescence microscope fitted with an AxioCam MR3 camera. Specific antigen detection by ELISA ELISA for polysaccharide detection AZD6244 manufacturer was CB-839 performed as previously described [17]. Briefly, antigens, bacterial cells or polysaccharide, were applied on a 96-well flat bottom high binding ELISA plate (Greiner) and incubated overnight at 4°C. Afterwards, plates were blocked by BSA and incubated with 20-kDaPS or PIA antisera for 1 h at 37°C. Peroxidase H-conjugated goat anti-rabbit IgG (Sigma Chemical Company, St

Louis, MO, USA), diluted 1:2,000 was added for 1 h. Color was developed by adding 100 μL/well SureBlue TMB Microwell Peroxidase Substrate (KPL). After incubation for 15 min at room temperature in the absence of light, the reaction was terminated with 100 μL/well of 1 M H2SO4 and measured at absorbance450. ELISA was also performed, as previously described, on 96-well tissue culture plates (Nunc) with similar Cyclin-dependent kinase 3 results. PIA isolation Isolation of PIA antigen was performed, as previously described [6], with slight modification. Briefly, S. epidermidis 1457 was grown for 22 h at 37°C with shaking at 100 rpm/min in 900 mL of TSBdia, prepared by dialysis of 100 mL of 10-fold-concentrated TSB against 900 mL of water. Bacterial cells were collected by centrifugation and were suspended in 20 mL of PBS. The antigen was extracted by sonicating cells four times for 30 sec on ice (Branson Digital Sonifier). Cells were removed by centrifugation at 6,000 rpm for 30 min at 4°C, and extracts were clarified by centrifugation for 60 min at 12,000 rpm. The extracts (20 mL) were filter sterilized, dialyzed against 50 mM Tris–HCl, pH 7.