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25 ��L of SYBR?-Green Master Mix (Applied Biosystems, Foster City, CA, USA), 12.5 ng of template cDNA and 400nM each primer in a final volume of 12.5 ��L. All amplicons showed the correct sizes after gel electrophoresis and the dissociation curves showed one distinct melting peak, ensuring the absence of a non-specific byproduct or primer dimers. Moreover, no reverse transcription controls and no template controls were taken, and the difference between the samples was at least eight ct-values, but most of the times >40 cycles (data not shown). Table 1. Sequences of mouse primers used for real-time qPCRa Immunohistochemistry UNC2881 Immunohistochemical staining was performed on 7-��m cryosections PLP-fixed kidney samples. Sections were stained with guinea pig anti-TRPV5 [25], rabbit anti�Ccalbindin-D28K (Swant, Bellinzona, Switzerland) and rabbit anti-NCC [26], as described previously [27, 28]. Images representing the entire kidney cortex were made using a Zeiss fluorescence microscope (Sliedrecht, the Netherlands) equipped with an AxioCam digital photo camera. For semi-quantitative determination of protein levels as the mean of integrated optical density [28], images were analysed with the Fiji ImageJ image analysis software (http://pacific.mpi-cbg.de). Immunoblotting Kidneys of control and furosemide-treated mice were homogenized in homogenization buffer A (HbA; 20 mmol/L Tris/HCl buy STI571 (pH = 7.4), 5 mmol/L MgCl2, 5 mmol/L NaH2PO4, 1 mmol/L EDTA, 80 mmol/L sucrose, 1 mmol/L phenylmethanesulfonylfluoride, 1 ��g/mL leupeptin and 10 ��g/mL pepstatin). Protein concentration of the homogenates was determined using the Bio-Rad Protein Assay (Bio-Rad, Munich, Germany). The proteins were solubilized by 30 min incubation at 37��C in Laemmli buffer. Sixty micrograms of each protein sample were separated on AP24534 molecular weight an SDS�CPAGE gel and blotted to a polyvinylidene difluoride�Cnitrocellulose membrane (Immobilon-P, Millipore Corporation, Bedford, MA, USA). Blots were incubated for 16 h with either a rabbit NCC antibody (1:500 dilution; Millipore, Billerica, MA, USA), a rabbit anti�Ccalbindin-D28K (1:10 000 dilution; Swant, Bellinzona, Switzerland), a rabbit anti-��ENaC antibody (1:1000 dilution; StressMarq, Victoria, Canada) or a mouse tubulin antibody (1:20 000 dilution; Invitrogen, Camarillo, CA, USA). Thereafter, blots were incubated with peroxidase-conjugated secondary antibodies after which proteins were visualized by chemiluminescence (Pierce, Rockford, IL, USA). Immunopositive bands were scanned using ChemiDoc XRS (Bio-Rad) and signals were analysed with the Quantity One software (Bio-Rad). The amount of NCC, calbindin-D28K and ENaC protein was normalized for the corresponding total amount of protein, using tubulin protein levels or Coomassie staining. Data are based on two independent experiments in which four samples per group were analysed. Statistical analysis Data are expressed as mean �� SEM. Statistical analyses were performed by Student's t-test. P