Figure 2. Quantification of SAA mRNA expression in response to UPEC. Four mice were either sham inoculated with PBS alone or infected with UTI89 via catheterization to initiate UTI. Alternatively, mice were infected systemically via IP injections of UTI89, with control animals recieving only PBS. After 1 d, levels of SAA1/2 and SAA3 in the (A) bladder wall, (B) isolated urothelium, and (C) liver were quantified relative to ribosomal L32 transcript levels. *, p,0.05 relative to sham Serum Amyloid A Induction by UPEC inoculated animals as determined by Student’s t test. The mean +/2 SD of triplicate experiments are shown. doi:10.1371/journal.pone.0032933.g002 and 14 days after instillation of UPEC were analyzed by a pathologist, provided no evidence of bacterial infection or inflammation. Urinary bladders, liver and blood were taken immediately after sacrifice. Intraperitoneal infections Four mice were inoculated via intraperitoneal injection with 200 ml of a suspension containing 108 CFU of UTI89 in PBS. The same amount of sterile PBS was received by four sham inoculated animals. After 1 day, mice were sacrificed and bladders, liver and blood were taken immediately. Immunofluorescence labeling Bladders were fixed for 2 h using 3% paraformaldehyde in PBS, rinsed overnight in 30% saharose, frozen, and cut into 7-mm-thick sections. After washing in PBS, sections were permeabilized in cold acetone for 5 min, quick-dried, blocked in 3% BSA in PBS and incubated overnight at 4uC with primary antibodies against SAA (rabbit polyclonal antibodies developed against synthetically produced peptides of SAA1/2; a gift of Prof. Malle, Medical University of Graz) diluted 1:100 in 1% BSA in PBS. After washing in PBS, samples were incubated in the dark at 37uC for 1 h with Alexa FluorH 555 labelled goat anti-rabbit secondary antibodies (Invitrogen, Molecular Probes, Leiden, Netherlands) diluted 1:300 in 1% BSA in PBS. After prolonged washing in PBS, sections were incubated at 37uC for 1 h with fluorescein isothiocyanate (FITC)-labelled rabbit polyclonal antibodies against E. coli (1:10; Abcam, Cambridge, UK). After additional washing in PBS, sections were mounted in antibleaching mounting medium Vectashield containing 49, 6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA) to stain DNA. Proper negative controls, in which both primary antibodies were replaced with serum from non-immunized animals, were per- formed. The samples were analysed using a Nikon Eclipse TE 300 fluorescence microscope (Amstelveen, Netherlands). ELISA Mouse serum was centrifuged from whole blood collected using a syringe in the heart, aliquoted, and stored at 220uC until used. The concentrations of SAA1 (Invitrogen, CA, USA) and SAA3 (Millipore, MA, USA) were measured in duplicate assays following the manufacturer’s instructions. Samples were thawed, allowed to reach room temperature and, if necessary, diluted with standard diluent buffer (1:40 for SAA1, undiluted for SAA3, except in samples from IP injected mice, where an 1:2000 was used) provided in the ELISA kits. After obtaining absorbance readings, SAA concentrations were calculated from standard curves. Statistical analysis was performed using mean 6 SD and Student’s t-tests. RNA isolation and quantitative PCR analysis Total RNA from various mouse tissues, including the liver, urothelium (mechanically peeled away from the bladder), and urinary bladder wall without the urothelium, was isolated using PureLink RNA Mini Kit (Invitrogen, CA, USA) following the manufacturer’s instructions. The purity and amount of RNA was determined by measuring the OD at a ratio of 260 to 280 nm. cDNA was generated from 1 mg of total RNA using the Reverse Transcription System (Promega, WI, USA) with oligo(dT) primers, and qPCR was performed with StepOne (Applied Biosystems, CA, PLoS ONE | www.plosone.org 3 March 2012 | Volume 7 | Issue 3 | e32933