Evos fl inverted microscope

To visualize ROS levels in the brains of study animals, 10 μm unfixed, frozen sections (midsagittal cut/plane) were processed through DHE staining using 150 μl of 10 μM DHE, slides were then cover-slipped and incubated at 37 °C for 45 min, as previously described by our laboratory (Lund et al., 2009 (link), 2011 (link); Oppenheim et al., 2013 (link)). Ethidium staining was visualized by fluorescent microscopy at 40 × on an EVOS FL-inverted microscope (ThermoFisher, Scientific, Richardson, TX, USA), digitally recorded, and analyzed by image densitometry (color images converted to white/black) using Image J software, from minimum of three slides, two sections each, three regions from each section (n = 3–4 per group). A subset of slides (two sections per slide, n = 3 per treatment/exposure group) were pretreated with flavoenzyme inhibitor, diphenyleneiodonium (DPI, 0.1 mM, Sigma, St Louis, MO, USA) in dark, moist chamber at 37 °C for 30 min prior to DHE staining.

Embryos were initially monitored twice daily following CRISPR injections. Embryos displaying divergent morphology were separated as “phenotypic” and surviving phenotypic embryos at 3 dpf were counted (Table 1). Subsequently, injected embryos were scored into three phenotypic classes under a dissecting microscope (Leica Zoom 2000) 24 hpf. Those with no apparent differences from wild-type embryos were scored into classification 1 (none). Those with abnormally small (or absent) heads and/or abnormally shaped eyes were scored into classification 2 (intermediate). Those with morphological abnormalities that prevented head and tail from being clearly distinguished were scored into classification 3 (severe). Representative embryos from each class are displayed in Figure S2.
For phenotypic characterization, live embryos were mounted 24 hpf in E3 medium (0.17 mM KCl, 0.33 mM CaCl₂, 0.33 mM Mg SO₄, 5% Methylene Blue) on a glass slide (Corning) with no coverslip and photographed under transmitted light on an EVOS FL inverted microscope (Thermo Fisher Scientific). Representative embryos were selected for imaging from total injected embryos (Table 2; N) Images were taken both in and out of the chorion. Knockout embryos were always imaged in conjunction with wild-type embryos from the same clutch.

ALI-COs were visualised on an EVOS FL inverted microscope (ThermoFisher) and using a microinjection capillary <0.2 μl of 1 mg/ml AlexaFluor 647-conjugate CTB (ThermoFisher, C34778) or 1-4 DiI crystals (ThermoFisher, D3911) were applied to the target region. DiI tracing was performed multiple times but due to difficulty with dye uptake and low signal-to-noise ratio only in one experiment was tracing of sufficient quality for analyis. Similarly, to achieve sparse neuronal labeling, ALI-COs were injected with <0.2 μl of CytoTune emGFP Sendai fluorescence reporter (ThermoFisher, A16519). Four days after CTB and DiI labeling and 5 days after viral emGFP labelling the samples were fixed for histological analyses.