Tcs sp5ii aobs confocal microscope

HeLa cells were seeded into 6-well plates, grown overnight, transfected with 1.5 μg of plasmid DNA, transferred on glass slides for microscopy twenty-four hours post-transfection, fixed in 4% paraformaldehyde and imaged forty-eight hours post-transfection. Subcellular localization of POU3F4 variants was determined by co-localization between wild type or mutant POU3F4 with EYFP fused to the N-terminus and 4’,6-Diamidino-2-Phenylindole (DAPI), as a marker of the nuclear compartment. Co-localization was detected and quantified as previously described (De Moraes et al., 2016 (link)). Shortly, imaging was performed by sequential acquisition with a Leica TCS SP5II AOBS confocal microscope (Leica Microsystems, Wetzlar, Germany) equipped with an HCX PL APO 63x/1.20 Lambda blue water immersion objective and controlled by the LAS AF SP5 software (Leica Microsystems). EYFP was excited with the 514 nm line of the Argon laser and emission was detected in the 525–600 nm range; DAPI was excited at 405 nm with a diode laser and emission was detected in the 420–485 nm range. Co-localization was quantified and expressed as the Pearson’s correlation coefficient (Adler and Parmryd, 2010 (link)), overlap coefficient and co-localization rate.

Cells were transferred to round 3-cm diameter glass slides 32 h post-transfection and fixed for 30 min with 4% paraformaldehyde in Hanks’ Balanced Salt Solution (HBSS) 48 h post-transfection. Imaging was performed in HBSS at room temperature. The FRET acceptor EYFP was excited with the 514-nm line of the Argon laser, and the emission was detected in the 525–600-nm range. The FRET donor ECFP was excited with the 405-nm laser, and the emission was detected in the 450–490-nm range. Imaging was performed by sequential acquisition with FRET AB-Wizard with a Leica TCS SP5II AOBS confocal microscope (Leica Microsystems, Wetzlar, Germany) equipped with a HCX PL APO 63x/1.20 Lambda blue water immersion objective and controlled by the LAS AF software (version 2.7.3.9723, Leica Microsystems). EYFP photobleaching in whole cells was obtained with 15 sequential illuminations at 514 nm (zoom factor 8×). FRET efficiency was calculated using the following formula:
ECFPpre and ECFPpost refer to the ECFP intensity before and after EYFP photobleaching, respectively, and were determined either in the bleaching regions of interest (ROI) or in plasma membrane ROIs, as indicated. In the latter case, ROIs were drawn within the bleaching ROI, on the pre-bleaching EYFP image, in areas with a clear targeting of αIIbs-EYFP at the cell periphery.

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.