Microseal a film

Leaf discs (7 mm) were made with a hole punch and placed on a 0.5% (w/v) agar pad in wells of a clear-bottom 96-well plate (Falcon) sealed with Microseal “A” film (Bio-Rad). The film over each well was punctured with a needle to allow for gas exchange. The prepared plates were dark-acclimated for a minimum of 6 h before placement in a BioTek Cytation 3 Imaging Reader. The baseline level of light transmittance through the leaf discs was calculated from measurements of absorbance values of 660 nm red light taken every 2 min for 20 min (red light does not activate chloroplast movement). To induce chloroplast movement in the cells, the plate reader was programmed to eject the plate for exposure to the selected light intensity for 2 min. The plate was then moved back into the plate reader for a recording of transmittance values (660 nm red light absorbance) for each well. After each reading, the plate was re-ejected to return to the blue-light treatment. The cycle of recording transmittance values and incubating with blue light was repeated for the indicated time periods for given light treatment. The calculated changes in light transmittance values were normalized to be relative to the starting “dark” position values.

At 10 dpf, morphologically normal larvae were removed from flasks using a sterile transfer pipet and placed into 48-well plates containing 500 µl of FS-10% HBSS per well. Plates were sealed with Microseal A film (BioRad, #MSA5001), wrapped in Parafilm, and placed in the dark in a temperature controlled behavior testing room at 26 °C, for at least 2 hr prior to testing. For testing, microtiter plates were placed on a Noldus tracking apparatus and locomotor activity was recorded for 40 mins. The light program consisted of a 20-min acclimation period in the dark (0 lux) followed by a 10 min light period (5 lux) and a 10 min dark period (0 lux). All tests were carried out between 11:15 am–4:00 pm. Videos were analyzed using Ethovision software Version 12 (Noldus Information Technology) as described previously^{48 (link)}.

Genomic DNA was prepared from individual whole zebrafish embryos or from fin-clips. Briefly, zebrafish embryos or fins biopsies were transferred into 96-well PCR plate containing lysis buffer (10 mM Tris-Cl, pH = 8.0; 50 mM KCl; 0.3% Tween 20; 0.3% NP40; 1 mM EDTA) plus proteinase K (3.4 mg/ml). The plate was sealed with film (Microseal “A” film, Bio-Rad, USA) and samples were digested at 50°C overnight in a humid chamber. Then, Proteinase K was inactivated by heating samples for 10 min at 95°C. The resulting genomic DNA was stored at -80°C and diluted in ddH₂O or 0.1xTE at proper ratio for PCR reaction.