Zyla 4

Zebrafish lenses were assessed for cataract at 4 days post fertilisation. Larvae were anaesthetised in tricaine methanesulfonate (300 mg/L, MS222 (Glentham Life Sciences), pH 7.0) and whole mounted in 0.5% w/v agarose and imaged using differential interference contrast illumination, under ×40 magnification, using an inverted microscope (Nikon Eclipse Ti-E) equipped with an s-CMOS camera, Zyla 4.2 plus s-CMOS camera (Zyla 4.2, Andor) and NIS-Elements AR acquisition software (Nikon).
Lens images from all experiments were collated, blinded and viewed with ImageJ [29 (link)] for cataract assessment by two independent researchers. Any divergent calls were resolved by a third researcher to obtain a result. Images were assessed for structural and organisational defects using a rubric of images. P values were calculated using a Fisher’s Exact test and experimental replicates were performed to confirm results. A post hoc power analysis (G*Power 3.1.9.4 [30 ]) from the aqp0a positive control experiments was used to determine that a sample size of >93 total embryos was required to achieve 80% power to detect an effect of size of 0.29, which is equivalent to the cataract rate of 16% observed with this morpholino in our laboratory.

Fluorescence
microscopy was performed at a custom inverted microscope described
in detail in a previous publication.^{31 (link)} Light
from a solid-state laser (561 nm, DPSS-System, MPB) was intensity-adjusted
using a half-wave plate and a polarizing beam splitter (WPH05M-561
and PBS101, THORLABS). The beam passed through a refractive beam-shaping
device (piShaper 6_6_VIS, AdlOptica) to create a flat illumination
profile. To achieve evanescent-field illumination, the beam excentrically
entered the oil immersion objective lens (100× NA 1.49 UAPON,
Olympus). Fluorescence emission was collected by the same objective
and filtered through suitable band-pass filters (605/64, AHF Analsentechnik)
before detection on a CMOS camera (Zyla 4.2, Andor). During acquisitions,
the temperature was stabilized at 23 °C (H101-CRYO-BL, Okolab),
and z-positioning of the sample was stabilized via
a piezo stage (Z-INSERT100, Piezoconcept and CRISP, ASI). The camera
was operated with the open source acquisition software μManager^{32 (link)} and images were acquired with 2 × 2 pixel² binning and field of view cropping to the central 700 ×
700 (prebinned) pixels to achieve an effective pixel width of 130
nm and a field of view matching the circular flat illumination profile
ca. 130 μm in diameter.

Cells were incubated with 5 μM fura-FFP18/AM for 2 h at 37 °C as described previously [45 (link)]. Coverslips with cultured cells were mounted on a perfusion chamber and placed on the stage of an epifluorescence inverted microscope (Nikon Eclipse Ti2, Amsterdam, The Netherlands) with an image acquisition and analysis system for videomicroscopy (NIS-Elements Imaging Software, Nikon). Cells were continuously superfused with HBS supplemented with 0.1% (w/v) BSA at room temperature and were examined at 40× magnification (Nikon CFI S FLUOR 40× Oil, Amsterdam, The Netherlands). Cells were alternatively excited with light from a xenon lamp passed through a high-speed monochromator Optoscan ELE 450 (Cairn Research; Faversham, UK) at 335 and 364 nm, and fluorescence emission, at 490 and 502 nm, respectively, was detected using a cooled digital sCMOS camera Zyla 4.2 (Andor; Belfast, UK) and recorded using NIS-Elements AR software (Nikon, Tokyo, Japan). Fluorescence ratio (F335/F364) was calculated pixel by pixel, and the data were presented as ΔF₃₃₅/F₃₆₄. TG-evoked Ca²⁺ entry was measured as the integral of the rise in fura-FFP18 fluorescence ratio for 3 min after the addition of extracellular Ca²⁺ taking a sample every second (AUC). To compare the rate of increase in fura-FFP18 fluorescence between different treatments, traces were fitted to the equation mentioned in Section 4.3.