Sutter dg 4 175w xenon arc lamp

Rats were anesthetized with isoflurane (2–5%) and isolation of mTAL tissue strips performed as described previously^{31 (link)}. Thin tissue strips containing mTALs were placed on a glass coverslip coated with the tissue adhesive Cell-Tak (BD Biosciences) for fluorescence imaging. Tissue strips containing mTALs were loaded with either dihydroethidium DHE (50 mmol/L) or 2',7'-Bis-(2-Carboxyethyl) -5-(And-6) - carboxyfluorescein (BCECF; 6 µmol/L) in HBSS for 1 hour at room temperature. Coverslips were placed on a heated imaging chamber maintained at 37°C (Warner Instruments) that allowed the rapid exchange of superfusion buffer and mounted on the stage of an inverted microscope (IX81 Olympus). mTAL were visualized with a ×40 water immersion objective lens. The signal was detected using a high-resolution digital camera (Photometrics Evolve, Roper Scientific). Excitation was provided by a Sutter DG-4 175W xenon arc lamp (Sutter Instruments) that allowed high-speed excitation wavelength switching. 3 to 10 mTAL epithelial cells were selected within each tissue strip to quantify changes in fluorescent intensity of dyes using Metafluor imaging software (Universal Imaging). Intracellular [Na⁺] was set at either 5 or 30mM using the same solutions as that used to set [Na⁺]_I in MØ.

Intracellular sodium concentration was measured using the sodium sensitive dye SBFI (Invitrogen) according to manufacturer’s instructions. In brief, 10nM stock solution of the dye in DMSO was further diluted in PBS (10ml) before loading onto the cells for 45 min. The signal was detected using a high-resolution digital camera (Photometrics Evolve, Roper Scientific). Excitation was provided by a Sutter DG-4 175W xenon arc lamp (Sutter Instruments). Regions of interest containing cells were selected using Metafluor imaging software (Universal Imaging). A FURA2 fluorescence lens kit was utilized to collect the fluorescent signal (Chroma Technology Corp, Bellows falls, VT). The ratio of fluorescent intensities at excitation 340nM/380nM/emission 510nM were recorded and the ratio calculated.