The administration of a single dose of LPS (10 mg/kg) as described.69 In short, mice had been offered a single intraperitoneal injection of either Escherichia coli LPS (10 mg/kg in 0.1 mL 0.9 standard saline) or 0.9 normal saline (controls). Mice were also provided 0.25 mL sterile saline as a series of subcutaneous injections every 12 h to reduce any contribution of volume depletion. Mice have been sacrificed at six, 24, or 48 h immediately after injection. The kidneys have been snap-frozen in liquid nitrogen and stored at -80 until extraction of total RNA or protein. For immunohistochemistry, kidneys had been promptly embedded in TissueTek OCT compound (Fisher Scientific), frozen, and stored at -80 . Analogous experiments were completed in which TNF- (R D Systems), dissolved in sterile PBS, was injected by tail vein into wildtype mice. Measurement of renal and blood parameters Blood was obtained at 2, six and 24 h just after TNF- was administered as a single i.v. dose of 0.5 or 2.five g. Blood and spot urine was obtained at 24 h just after LPS injection. TNF- levels were determined from sera obtained 2 h soon after TNF admistration utilizing a mouse TNF- ELISA kit in accordance with the manufacturer’s directions. (eBioscience, San Diego, CA). Enterokinase Protein MedChemExpress Plasma concentration of urea had been determined using a Beckman Coulter Synchron DXC600 autoanalyzer. Urine levels of albumin were determined using a commercially obtainable mouse albumin ELISA (Bethyl labs, Montgomery, TX). Urine levels of creatinine had been determined working with Enzymatic Creatinine LiquiColor?Reagent (StanBio Lab, Boerne, TX). Protein preparation and immunoblotting Frozen kidney tissue was thawed and homogenized for western blot as described.69 Membranes were incubated overnight with polyclonal rabbit antibodies against heparanase-1 and VEGF (Abcam, Cambridge, MA). Immediately after becoming washed, the membranes had been incubated for 2 h using the secondary antibody (800 nm goat anti-rabbit IgG, Li-Cor Biosciences, Lincoln, NE) along with the protein bands have been detected by an Odyssey infrared imager (Li-Cor Biosciences, ODY-1320). An actin handle was performed for every membrane. Band density was measured with ImageJ (v1.44p, NIH, USA) and normalized to actin for each lane. Immunofluorescence in kidney cryostat sections Cryostat sections (4 m) prepared from mice kidneys were fixed as described,69 and incubated at 4 overnight with key rabbit polyclonal antibody against heparanase-1, VEGFR2 (KDR antibody, Proteintech Group, Chicago, IL), or rat anti-Heparan Sulfate Proteoglycan (US Biological, Marblehead, MA), followed by incubation for 2 h at space temperature with secondary antibodies. Some cryostat sections immunostained as above were then either co-stained with rat antibodies for the endothelial marker VE-cadherinAuthor Manuscript Author Manuscript Author Manuscript Author ASS1 Protein Purity & Documentation ManuscriptKidney Int. Author manuscript; available in PMC 2014 July 01.Xu et al.Page(Abcam, Cambridge, MA) and CD31 (BD Bioscience, San Jose, CA), or with goat polyclonal antibody against nephrin (Santa Cruz Biotechnology, Santa Cruz, CA). For wheat germ agglutinin (WGA) staining, cryostat sections were incubated with Alexa Fluor 594conjugated WGA (Molecular Probes, Eugene, OR). The stained sections were then examined having a Fluoview 200 laser-scanning confocal microscope equipped with a 647-nm argon laser at ?0 and ?0 magnification. To quantify WGA expression, densitometric evaluation in the intensity in the fluorescence signals was performed on digitized images of glomeruli employing ImageJ software program (Na.
