Zen 2010 d software

Injected embryos (crispants) and non-injected siblings (controls) were raised in 90 mm petri dishes (approximately 50 embryos per dish) containing 25 mL of embryo medium. Between 24–26 hpf, all crispant and control embryos were dechorionated by a gentle 20 min treatment with pronase (0.6 mg/mL). Thyroid development was monitored in live zebrafish at 55 hpf, 80 hpf and 6 dpf by visual inspection using a M165 FC fluorescence stereomicroscope (Leica). For the duration of the phenotypic analysis, embryos and larvae were anesthetized in 0.02% tricaine. All control and crispant fish were inspected for gross developmental defects, deviations in size, shape or location of the thyroid, and for the overall intensity of the fluorescent reporter signal.
For documentation of thyroid phenotypes, embryos and larvae were immobilized in 1% low-melting point agarose (Lonza) containing 0.016% tricaine and imaged on Fluoro-Dish glass bottom dishes (WorldPrecisition Instruments). Images of the ventral pharyngeal region were acquired using a DMI600B epifluorescence microscope equipped with a DFC365FX camera and LAS AF Lite software (Leica). If indicated, confocal live imaging was performed with a LSM 510 confocal microscope (Zeiss) using Zen 2010 D software (Zeiss).

Immuno-staining for STORM imaging were performed as described above for confocal imaging. In addition paraformaldehyde was quenched with 50 mM NH₄Cl for 10min immediately after fixation. Cells were stained with Cav1 and Alexa Fluor 647 secondary antibodies and washed with PBS five times for 15 minutes per wash on a rocker.
dSTORM images were acquired with the same instrument as described for PALM. In order to excite and switch-off the fluorescent dye, Cav1-Alexa flour 647 was excited and switched off with the 642-nm excitation laser at maximum power (150 mW). Once the excited molecules were switched off the excitation laser was reduced to 45 mW and 20,000 frames (exposure time 30 ms and gain 50 V) were acquired in order to obtain super-resolution images. During acquisition, the 405-nm laser (between 0.05–2.5mW) was used to further activate more molecules when blinking rate was low.
The composition of the STORM buffer used for Alexa Fluor 647 includes: 25 mM HEPES, 25 mM glucose, 5% glycerol, pH 8.0, 10 mM 2-Mercaptoethylamine (SIGMA #30070), 50 μg/ml glucose oxidase (SIGMA #G2133) and 25 μg/ml of horseradish peroxidase (SIGMA #P8375). As with PALM, drift correction was performed with fiducials and the Zeiss software Zen 2010D software. dSTORM data analysis was performed as described for PALM data analysis.

Confocal microscopic analyses of the thyroid region of zebrafish embryos were performed on vibratome sections of WIF-stained specimens. After WIF staining, zebrafish embryos were embedded in 7% low melting agarose and cut into serial 100 µm thick sections on a vibratome. Sections were mounted in Glycergel (Dako) on custom-made #1.5 72×26 mm glass slides. Confocal images of zebrafish sections were acquired using an LSM510 confocal microscope (Zeiss) and Zen 2010 D software (Zeiss). For quantitative analyses of cell numbers, confocal z-stacks covering the whole thyroid region were acquired and the number of cells expressing specific markers was determined manually by analyzing consecutive z-stacks. Images were processed using ImageJ/Fiji with a 1-pixel radius minimum filter to remove artefacts.