To begin, use an incubator and an LED Microplate Illuminator to create a light box for controlling light exposure and temperature. Then, position a six well plate on the upper shelf of the incubator, and check whether the light beam evenly encompasses it. Use a sheet of paper to visualize the distribution of light coverage.
Use a light meter to measure irradiance and spatial uniformity. At least one day before injections, make injection dishes and agarose-coated six well plates. Rinse an injection dish mold with embryo medium, and place gently onto molt and agarose.
Once the agarose has solidified, delicately use the tab to remove the mold. Next, prepare flamed glass pipettes for immunofluorescence experiments on dechlorinated embryos. Place the end of a glass pipette into a bun and burner flame, and rotate continuously until the edges become smooth.
Prepare an injection mix using the mRNAs shown here. For the phenotyping assay, introduce the needle through the corion directly into the center of the cell at the one cell stage, and inject one nanoliter of the injection mix. If conducting an immunofluorescence assay, target the center of the cell of coated embryos at the one cell stage.
For both assays, prepare enough embryos for the unexposed non-injected, unexposed injected, exposed non-injected, and exposed injected embryos. Select at least 30 embryos per condition. For the immunofluorescence assay, prepare an additional dish containing non-injected embryos as a proxy to assess the progression of developmental stages.
After injection, incubate the embryos in Petri dishes at 28 degrees Celsius.
