Normal goat serum (Jackson ImmunoResearch Laboratories) in PBS for 30 min at room temperature. After washing three times with PBS for 5 min, the samples were incubated for 3 h at room temperature in the dark with anti-TRPV4 antibody (1:1000 dilution; Alomone Labs) in 1 normal goat serum and 0.1 Triton X-100 in PBS. Subsequently, the samples were washed three times with PBS and incubated for 1.5 h at room temperature in the dark with goat anti-rabbit IgG labeled with Alexa Fluor 488 (1:1000 dilution; Invitrogen) in 1 normal goat serum and 0.1 Triton X-100 in PBS. After washing three times with PBS for 5 min, the samples were stained with DAPI (1.5 M; Calbiochem) to visualize nuclei. Subsequently, the samples were dehydrated and mounted with permanent mounting medium (Thermo Scientific). Labeled tissue samples were examined with an Nikon Eclipse Ti inverted confocal fluorescence microscope using a 40 Plan Fluor oil immersion (1.Colesevelam (hydrochloride) 3 numerical aperture) objective. Samples were excited with 405 and 488 nm laser diodes, and emission was captured with a 16-bit CoolSNAP HQ2 camera (Photometrics) interfaced to a PC running NIS-Elements version 4.00 software. Three-dimensional stacks of splitopened distal nephrons were generated from a series of confocal plane images with 0.25- m steps. Solutions–The typical bath solution was 150 mM NaCl, 5 mM KCl, 1 mM CaCl2, 2 mM MgCl2, 5 mM glucose, and 10 mM HEPES (pH 7.4).(-)-Epicatechin All reagents were applied by perfusing the experimental chamber at 1.5 ml/min. To test the effect of elevated flow on [Ca2 ]i, the rate of perfusion was instantly increased from 1.5 ml/min ( 15 mm H2O) to 15 ml/min ( 80 mm H2O). Using a parallel plate chamber, we recently estiJOURNAL OF BIOLOGICAL CHEMISTRYEXPERIMENTAL PROCEDURES Materials and Animals–All chemicals and materials were from Sigma, VWR International (Radnor, PA), and Tocris Bioscience (Ellisville, MO) unless noted otherwise and were reagent grade. Animal use and welfare adhered to the National Institutes of Health Guide for the Care and Use of Laboratory Animals following a protocol reviewed and approved by the Institutional Laboratory Animal Care and Use Committee of The University of Texas Health Science Center at Houston.PMID:23310954 For experiments, 6 8-week-old C57BL/6 mice (Charles Rivers Laboratories, Wilmington, MA) were used. Animals were maintained on standard rodent regimen (Purina 5001) and had free access to tap water. Tissue Isolation–The procedure for isolation of the collecting ducts and the connecting tubules from C57BL/6 mice suitable for Ca2 imaging and immunofluorescence microscopy closely follows the protocols reported previously by us (20 3). Kidneys were cut into thin slices ( 1 mm) and placed into an ice-cold bath solution buffered with HEPES (pH 7.4). Distal nephrons were visually identified by their morphological features (pale color, coarse surface, and, in some cases, bifurcations) and were mechanically isolated from kidney slices by microdissection using watchmaker forceps under a stereomicroscope. Isolated distal nephrons were attached to 5 5-mm cover glasses coated with poly-L-lysine. A cover glass containing a distal nephron was placed in a perfusion chamber mounted on a Nikon Eclipse Ti inverted microscope and perfused with bath solution at room temperature. Distal nephrons were split open with two sharpened micropipettes, controlled with different micromanipulators, to gain access to the apical membrane. The nephrons were used within 1 h of isolati.