Las af sp5

Sections were examined with a Leica TCS SP5 confocal laser-scanning microscope (Leica Microsystems, Mannheim, Germany) using a sequential scanning procedure and, when appropriate, an overexposed laser setting. Confocal images were taken at 0.35 µm intervals through the z-axis of the sections, with 40× and 63× oil lenses associated to zoom factors from 1.5 to 3. Single, serial optical planes and z-stacks (projection images) were analyzed by Leica confocal software (Multicolour Package; Leica Microsystems). The size of TNT-like structures was evaluated with LAS-AF SP5 software (Leica Microsystems) on 63× magnification fields zoomed 3 times. TNT thickness (µm) was measured on projection images from fetal cerebral cortex (n = 4), stained for NG2, for a total of 63 TNT fields. The results are expressed as mean ± standard deviation (M ± SD) together with the maximum (Max) and minimum (Min) values.

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.

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.