Quantitative Imaging of Pendrin Expression

Quantitative imaging was performed as formerly described [28 (link),29 (link)]. Shortly, cells expressing the fusion proteins SLC26A4-EYFP or SLC26A4-EYFP were fixed with 3% paraformaldehyde for 30 min, counterstained with 0.1 μg/mL 4′,6-diamidino-2-phenylindole (DAPI) for 10 min, thoroughly washed, and imaged in Hank’s balanced salt solution (HBSS, Sigma-Aldrich). Imaging was performed with a Leica TCS SP5II AOBS confocal microscope (Leica Microsystems, Wetzlar, Germany) equipped with a HCX PL APO 63×/1.20 Lambda blue water immersion objective and controlled by the LAS AF SP5 software version 2.7.3.9723 (Leica Microsystems). EYFP was excited with the 514 nm line of the Argon laser, and emission was detected between 525 and 600 nm; DAPI was excited with a diode laser (405 nm), and emission was detected between 430 and 470 nm. Laser power and photomultipliers gain were kept rigorously constant for the acquisition of all images. To obtain pendrin expression levels normalized for the cell density, the fluorescence intensity (in average levels of gray) of the whole imaging field in the EYFP emission window was subtracted for the background fluorescence and normalized for the background-subtracted fluorescence intensity in the DAPI emission window.