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    Day Before Microinjection

    1) The day before you want to have eggs ready for microinjection (3:00pm-6:00pm) set up at least 4 breeding bins. To ensure that you'll definitely get eggs, i suggest setting up all 8 breeding bins. Each bin will have two females and one males. The first time you attempt setting up breeding bins do it with Amy's help. She will explain the difference between male and female. But in a few words, male zebrafish are distinguished by the yellow shade of the abdomen and its straight and flat nature, while the female zebrafish’ abdomen tends to be more rounded.
    2)Make sure that the air tank attached to the picospritzer, the picospritzer itself, and the microinjection are working properly and are ready to be used tomorrow for the microinjections. Otherwise, prepping it will take up time the morning of microinjections. Also, make sure to have prepared at least 3-4 microinjection needles the day before. The microinjection needles can be made using the needle-puller which should have all three dials set to 999.

    Day of Microinjection

    3) The day of microinjection you need to move the zf from their room to the lab soon after the lights are on in their room. Ask Amy for the exact time the lights turn on but usually between 7:30 am 8:00am.
    4) When the bins with the zf have been moved to the lab, move the agarose microinjection mold, the Isl2b plasmid, the phenol red, the microinjection needles, and the micro scissors to the microinjection station. Turn on the picospritzer and the air tank.
    5)Move the agarose mold under the microscope. Load the microinjection needle with 2ul of the Isl2b plasmid and 1ul of the phenol red. Once loaded, insert to the needle into the picospritzer. Under the microscope, break the end of the needle with the help of the micro scissors. It is crucial for the microinjection to be successful to have the tip correctly broken. Too thin and long needles do not easily penetrate the embryo’s chorion, while too short and thick ends microinject too much.
    6) When the needle is ready the bin dividers can be removed and the zf allowed to breed. REMEMBER, steps 3-6 need to happen within the first 45 minutes after the lights are on in the zf room downstairs. If you allow more time than that then the chances of getting zf embryos decrease dramatically.
    7) Check the bins regularly (every 5 minutes) after the dividers are removed. Once you see a bin with eggs, immediately transfer the eggs into the agarose mold. Align the eggs in the ridges and then slowly remove the excess liquid from the mold. The more the liquid the more unstable the eggs will be and harder the microinjections.
    8) Set up the picospritzer so the liquid coming out of the needle is not bigger than 1/4 of the size of the eggs. Do the microinjections, keeping track of the eggs you’ve already microinjected.
    9) Once microinjected, transfer the eggs to a separate petri dish and fill it with liquid from the bins or with 1X E3 media. Transfer the petri dish to the zf incubator.

    3.5 days after Microinjection (~84hours)

    10) Anesthetize the zf that survived the microinjection in tricaine solution.
    11) Once anesthetized, move the in fix vials. Remove any excess tricaine solution left in the vial. Fill in the vial with BT Fix.
    12) Leave the zf in the BT overnight. Then wash 3x for 5 minutes with Fix PBS.

    Flatmounting - Within maximum 1 week after fixation

    13) Place one zebrafish a glass slide making sure that the amount of liquid transferred along was appropriate. Too much liquid hinders the dissection of the retina, whereas too little liquid dries out quickly which could be detrimental in the flatmount preparation.
    14) The zebrafish retinae are both removed from the rest of the body using both a pair of forceps and a fine scalpel blade. Once the retinae are carefully cut into smaller pieces and then placed with the inner part of the retina pacing upwards.
    15) When most of the pieces of the retinae are facing the right way, you should wait for the excess liquid to evaporate but also make sure that the pieces of retinae are not drying out and then coverslipped using a small drop of gel mount.
    16) Repeat for all the zf in the vial.
    17) Line the edges of the coverslip with nail polish to prevent evaporation of the liquid. Label and let at least 6-10 hours before analyzing them under the microscope

    Analyzing the z-stacks
    When tracing out the dendrites from the z-stacks, I suggest you zoom in at 200% but keep that change consistent for all your tracings. It makes tracing a lot easier and the dendrites much clearer.