System. Dilated renal pelvis was located with Vevo 770 system and the RMV-704 scanhead. Syringe with 30 gauge fine needle was used to quick penetrate abdominal skin and reach to the dilated renal pelvis under the guidance of ultrasound scan. After the needle tip was visible inside the renal pelvis, urine was withdrawn through the syringe until the hydronephrosis disappeared. The needle was then quickly withdrawn out of the skin. Histology analysis of mouse embryos, kidneys, and ureterovesical junctions The mouse embryos bodies, mouse kidneys, or ureterovesical junction regions were dissected and fixed in 4% paraformaldehyde overnight, and taken through a graded alcohol series for paraffin embedding. The paraffin blocks were sectioned at 4 mm using a MT-920 microtome (MICROM) and stained with standard Hematoxylin/Eosin method. The histology slides were evaluated under an Olympus BHT light microscope equipped with a SPOT digital camera system. Tissue in situ hybridization In situ hybridization analysis was performed with digoxigenin- labeled Robo2 and Slit2 riboprobes as previously described [18,59]. The Robo2 and Slit2 cDNA were linearized with NotI and probes were generated using the DIG DNA labeling and detection kit (Roche Applied Science). Hybridizations were performed on 4% paraformaldehyde (PFA) fixed OCT embedded mouse embryonic urinary tract frozen sections. Briefly, mouse embryos were collected and dissected in ice-cold PBS and fixed overnight at 4uC in 4% PFA in 0.1 M phosphate buffer and embedded in OCT. Frozen sections were cut on Cryostat and air-dried for 1– 3 hours, and fixed again in 4% PFA for 10 minutes at room temperature. After washing with PBS three times, sections were treated with proteinase K (1 mg/ml for 3 minutes at room temperature), washed three times with PBS, acetylated for 10 minutes at room temperature and washed again with PBS. Prehybridizations were performed for 2 hours at room tempera- ture in a humidified chamber. Hybridizations were performed at 68–72uC overnight in a humidified chamber. Prehybridization and hybridization solution were made as following: 50% formamide, 56 SSC, 56 Denhardts, 250 mg/ml baker’s yeast RNA, 500 mg/ml herring sperm DNA. Washes were performed at 72uC in 56 SSC for 5–10 minutes, then at 72uC in 0.26 SSC for 1 hour. Sections were stained overnight with anti-digoxigenin antibody (Roche Applied Science) at a 1:5000 dilution in buffer with 0.1 M Tris-HCl, pH 7.5, 0.15 M NaCl, and 1% heat- inactivated goat serum. After staining at 4uC overnight in a humidified chamber, slides were washed in solution with 0.1 M Tris-HCl, pH 7.5, 0.15 M NaCl. Alkaline phosphatase activity was then detected by developing slides in BCIP, NBT (Roche Applied Science) and 0.25 mg/ml levamisole in a humidified chamber for one day in the dark. Sections were dehydrated and mounted in Permount (Fisher Scientific) and photographed under microscope. Early mouse embryonic kidney organ culture and cytokeratin straining Mouse metanephric embryonic kidneys were mechanically dissected from E11.5 Robo2del5/del5 homozygous and control embryos as previously described [60]. Briefly, embryonic kidney tissues were dissected from E11.5 mouse embryos in ice-cold CO2- independent DMEM medium (Invitrogen) or Dulbecco’s PBS (Ph 7.2–7.4, with Ca2+ and Mg2+) containing 10% fetal bovine serum (Invitrogen) using two pairs of fine-tip forceps under dissecting Antenatal Hydronephrosis and VUR in Robo2 Mice microscope. The isolated embryonic kidney explants were transferred to 12-well Transwell tissue culture plates (Costar #3401) and were placed at the medium-air interface of Transwell polycarbonate membrane filter (0.4 mm pore size). Each individual well within the 12-well Transwell tissue culture plate contains 800 ml DMEM/F12 culture medium (Invitrogen). The kidney explants were cultured for 24 hours at 37uC in tissue culture incubator with 5% CO2 and then fixed in 100% methanol at 220uC for 15–30 minutes. The fixed kidney tissues were washed in 16 PBST for 5 minutes and incubated with anti-pan- cytokeratin primary antibody (Sigma, #C9687, 1:50 dilution) at 37uC for 1–2 hours. The tissues were then washed in 16 PBS for 5 minutes at room temperature and strained with Cy3 conjugated secondary antibody (Jackson ImmunoResearch, #715-165-150) in a 1:50 dilution at 37uC for 1 hour. The stained kidney explants were washed in 16 PBS and mounted in VECTASHIELD mounting medium (Vector Laboratories, #H-1000). Ureteric buds were examined and photographed using a Perkin Elmer Ultra- View LCI multi-point spinning disc laser-scanning confocal microscope. Dual and triple immunofluorescence staining of mouse embryonic ureter Dual and triple immunofluorescence staining were performed on E14.5 Robo2 mouse embryonic ureter tissues which were dissected and fixed in 4% paraformaldehyde (PFA) at 4uC for 2 hours. The tissues were washed with 16 PBS and followed by two 30-minute incubation in 15% and 30% sucrose for cryoprotection. The fixed embryonic ureters were frozen embedded in OCT compound and sectioned at ,8 mm thickness using Cryostat. Sections were washed in 16 PBS buffer and incubated in 16 casein blocking solution (Vector Laboratories, #SP-5020) for 30 minutes at room temperature. Sections were then stained with a monoclonal anti-a smooth muscle actin (anti- aSMA) antibody (clone 1A4, Sigma-Aldrich, # A5228) at 1:200 dilution in 16 casein blocking solution at 4uC overnight. For double staining with E-cadherin, a goat-anti-mouse E-cadherin primary antibody (R&D Systems, #AF748) was also added to the sections at 1:100 dilution. After three times 5-minute washes with 16 PBS buffer, sections were incubated in a Cy3 conjugated secondary antibody (Chemicon, #AP192C, 1:400 dilution) and a FITC conjugated secondary antibody (Jackson ImmunoResearch, #705-095-003, 1:100 dilution) for 4 hours at 4uC followed by 15 minutes at room temperature. For lectin staining, the ureter sections were incubated with FITC-conjugated urothelium marker lectin Dolichos biflorus (DBA, Sigma-Aldrich, #L9142) 4 hours at 4uC followed by 15 minutes at room temperature. After three times 5-minute washes in 16 PBS, dual-stained sections (anti-aSMA and FITC-conjugated DBA) were mounted with regular VECTASHIELD mounting medium (Vector Laboratories, #H-1000) and triple-stained sections (anti-aSMA plus anti-E-cadherin) were mounted with modified VECTA- SHIELD mounting medium with DAPI (Vector Laboratories, #H-1200). Stained sections were examined and photographed using a Perkin Elmer UltraView LCI multi-point spinning disc laser-scanning confocal microscope. Statistics and mouse survival curve analysis The renal pelvic diameters in Robo2 mice with progressive hydronephrosis were analyzed with Student’s t-test. The survival curve of Robo2 mice with antenatal hydronephrosis was analyzed with NCSS 2007 software (Kaysville, Utah). PLoS ONE | www.plosone.org 11 September 2011 | Volume 6 | Issue 9 | e24763