Dmi6000 b system

Cell imaging was performed at room temperature with automated widefield epifluorescence microscopes. With a Scan̂R microscope (Olympus Biosystems)46 (link) equipped with a metal halide light source (MT20), a 12 bit 1344 × 1024 pixel resolution C8484 CCD camera (Hamamatsu) and a 10× UPlanApo objective (Olympus) with a numerical aperture of 0.4. Exposure times at maximum light brightness for Hoechst, mCherry and Alexa Fluor® 647 were 10–20 ms, 450 ms and 850 ms (wt-CFTR) or 15–30 ms, 700 ms and 1000 ms (F508del-CFTR), respectively. The Hoechst channel was used for contrast-based autofocus. Fluorescence images in Fig. 2 were acquired with a Leica DMI6000 B system equipped with a metal halide light source (EL6000) and a DFC365 FX CCD camera (Leica) with a 1392 × 1040 pixel resolution and 12 bit grayscale representation. A 10× HC PL APO objective (Leica) with a numerical aperture of 0.4 was used.

Light microscopy was performed with the Leica DMI6000 B system to monitor the surface and cortical changes following the insemination of immature oocytes and mature eggs in acidic or alkaline NSW at different time points. For TEM analyses, immature oocytes and oocytes matured and fertilized in acidic or alkaline seawater were fixed in NSW containing 0.5% glutaraldehyde (pH 8.1) for 1 h at room temperature. After extensive washing in NSW, the samples were post-fixed with 1% osmium tetroxide and 0.8% K₃Fe(CN)₆ for 1 h at 4 °C. The samples were washed in NSW and rinsed with distilled water (3 times, 10 min each), and finally treated with 0.15% tannic acid for 1 min at room temperature. The specimens were then dehydrated in ethanol with increasing concentrations. Residual ethanol was removed with propylene oxide before embedding in EPON 812. Ultrathin sections were made with the ultramicrotome Leica EM UC7 (Leica Microsystems, Wetzlar, Germany) and observed under a Zeiss LEO 912 AB (Carl Zeiss Microscopy Deutschland GmbH, Oberkochen, Germany) without staining.