Inducible expression of cyfip2-EGFP, as well as C179R and K723E variants, was initiated at 30 hpf by placing dechorionated larvae into 96-well plates and incubating at 38°C for 15 or 40 minutes [32 (link)]. Following heatshock, larvae were returned to Petri dishes, and given 4 days of recovery at 29°C. GFP fluorescence was confirmed between 4–6 hours post-heatshock for startle experiments using a Nikon SMZ25 stereo microscope with a GFP bandpass filter and Lumen 200 fluorescence illumination system. For day 5 heatshock rescue experiments, larvae were given 4 hours of recovery at 29°C prior to startle sensitivity testing.
For imaging experiments, larvae were treated as above for transgene expression at 30 hpf and at 29°C for 1 hour recovered in petri dishes in groups ≤ 65. After 1 hour of recovery fluorescence was verified, and larvae were visualized using the stereo microscope system described above and larval images were captured at 1-, 3-, 6-, 18-, 24-, 30-, and 42-hours post-heatshock using a Nikon DS-Qi2 monochrome microscope camera. Image analysis was conducted using FIJI (ImageJ) analysis software to manually define ROIs encompassing the entire larval body, excluding the eye and auto fluorescent yolk sac. Fluorescence intensity values reflect the mean gray values recorded for respective ROIs.
For imaging experiments, larvae were treated as above for transgene expression at 30 hpf and at 29°C for 1 hour recovered in petri dishes in groups ≤ 65. After 1 hour of recovery fluorescence was verified, and larvae were visualized using the stereo microscope system described above and larval images were captured at 1-, 3-, 6-, 18-, 24-, 30-, and 42-hours post-heatshock using a Nikon DS-Qi2 monochrome microscope camera. Image analysis was conducted using FIJI (ImageJ) analysis software to manually define ROIs encompassing the entire larval body, excluding the eye and auto fluorescent yolk sac. Fluorescence intensity values reflect the mean gray values recorded for respective ROIs.
