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    • CommentAuthornameyer
    • CommentTimeAug 1st 2007 edited

    This protocol explains how to: mount and image live zebrafish

    Protocol revised: 8/1/2007

    Protocol written by: Natasha Meyer

    Before Imaging (mounting the fish)

    NOTE:  I changed my solutions everyday of the experiment to ensure that the malathion wouldn't degrade.

    1. Screen your fish for GFP before putting them in agar -- it will save you time when you're imaging. 
    2. Make a stock solution of 0.4% fish anesthesia (tricaine / MS222 in E3) with a pH between 7.2 and 7.4. I filled two 50 ml Falcon tubes - 0.4 g of tricaine dissolved in 100 ml of E3). Keep stock in the freezer.
    3. Make up 500 ml of 0.016% tricaine in E3 (you can use your stock and dilute it or dissolve 0.08 g in 500 ml and then pH it). Keep 0.016% tricaine in the refrigerator. 
    4. Dechorinate the fish using Pronase or forceps. Alternatively, you can dechorinate them the night beforehand to save time in the morning.
    5. Place embryos in 0.016% tricaine in their respective dishes (use the BD Falcon p35 dishes - they fit into the plexiglass holder you'll be using to image.  Place 4-5 embryos in each dish).
    6. Place your 0.4% frozen stock solution in a water bath @ ~ 150 C.
    7. Make 1.5% low-melting-point agarose in E3. If you're only doing about 6 small plates, use 0.15 g of low-melting-point agarose in 10 ml of E3. Heat it in the microwave for about 15 seconds - watch it to make sure it doesn't bubble over.
    8. Place agarose in the water bath to let it cool down but not harden.
    9. Add 400 ul of the 0.4% tricaine stock to the 10 ml of agarose to make it a 0.016% tricaine/agarose solution. (Make sure to add the tricaine after heating the agarose).
    10. Remove excess Tricaine from the Petri dishes - leaving enough to cover all of the fish.
    11. Pour the agarose into the dish until about half of the surface is covered. Swirl the dish to spread the agarose.
    • Make sure you don't have too much agar in the bottom of the dish. The objective lens will hit the bottom when it's trying to focus through the agarose and end up killing the fish. In this case, less is better.
    • Make sure your agar isn't too hot or it can kill the fish. Wait at least a minute after microwaving.
    1. Use a pipette and pick up the embryos which will have floated to the sides of the dish. Pipette them back into the center.
    2. Under the microscope, line up the embryos and position them as desired using insect pins (I suggest having one straight insect pin to drag them and one L-shaped pin to straighten their bodies out).
      • If the embryos are too curved to lie flat in the agarose, try dechorinating them earlier.  I had mixed results using the pronase to dechorinate the fish at 24 and 48 hpf.  They didn't curl as much, but more of them died.  I found dechorinating the night before I imaged to be helpful because they were flatter and easier to mount, even the deformed ones.
      • If you have fish in a straight line, they will be simpler to image.   You can remain on the 60x lens and just move up or down the row (instead of having to switch to the 4x between each image to find the next fish).
    3. Let the agarose cool for about a minute before picking the dish up. The embryos may shift positions if the agarose isn't solid.
    4. When the agarose solidifies, add some 0.016% Tricaine on top to keep the agarose moist. Put it on gently at first around the embryos otherwise you could push them out of the agarose. If you are using the 60X water objective on the confocal microscope, you will want to fill the dish all the way to the top with liquid.  


    While Imaging:

    1. It takes about 2 and a half to 3 hours to image 12 fish, so plan accordingly.  Put your p35 dish with the lined embryos into the plexiglass holder to put it on the scope.
    2. To find the embryos on the confocal microscope, focus with the 4X objective. Center the eye of the first embryo in your field of view.
    3. Use the coarse focus to move the stage as far down as possible and switch over to the 60X objective. Slowly raise the stage with the coarse focus while looking into the eyepieces. When the objective hits the water, the view lightens. All of the sudden the eye should pop up at you.
    4. Once you start to make out the eye or a part of the fish, switch to fine focus. If it starts to get dark again, you've probably passed the point of focus. Look slightly to the left and right to try and find the embryo.  When you've focused, take a brightfield image of the embryo.
    NOTE: If you're trying to capture a brightfield image on EZ-C1, make sure you have the detector set to "small," gain 4 pretty high, and that the color selected for C1-Live:1 is gray.  You also need to have the  488 laser power up and not the 633. Finally, save it as a .BMP!! You can't see it otherwise.
    1. After you've taken a brightfield image with EZ-C1, you can just turn the correct gain up, change the detector size and focus to find the neurons. You can put the gain up very high to find them and then just decrease the gain to the level you want to take pictures at.


    • The 60X water objective doesn't focus well unless your dish has a lot of liquid.  I kept a 15 ml falcon tube filled with 0.016% tricaine in the confocal room and changed it out every week. It probably wouldn't work well as anesthesia (tricaine degrades) but is convenient when you need to add liquid to your petri dish.  You could keep distilled water on hand to fill the dishes to the top.
    • When imaging the whole fish for GFP, make sure the detector is open - you'll get clearer pictures and if you only have 4 or 5 embryos on a dish you won't bleach them out.
    • If your embryo is not quite straight, you can rotate the stage by pushing on the bottom. Be careful not to move the stage up or down while you’re rotating; you'll lose your location.
    • I suggest imaging all the way to the top and bottom of the fish.  You'll get ugly renderings with melanophores all over the place, but you can crop it out later to get prettier pictures.
    • When you're ready to use the coarse focus to move the stage down and switch dishes, make sure EZ-C1 is closed. Otherwise the microscope will not let you coarse focus all the way down (as a safety precaution).    


    After imaging:

    With regards to the embryos:

    1. Use a pipette with a smaller tip and suck up some of the liquid. Squirt the liquid back over one of the embryos heads. Continue to do this until the embryos wriggles free. 
    2. Remove all of the embryos and fix them or euthanize them.

    With regards to your images:

    1. You can crop out sections of the .avi files using ImagePro. Watch the video and determine which frame should be your first and which should be your last.
    2. Open the video file again and select "Load Selected Portion." Put in the starting frame # and input the final frame #.   In the "number of frames" box, put in the first frame #.  
    3. Click OK and save the new file as another .avi file.
    4. Go back to EZ-C1 in the confocal room and open your cropped files. Select Data > Volume > Volume Render.  (I used this cropping procedure for specific axons, so I could tell what belonged to what)
    5. The pictures might come out red. If  you need them to be green go into Photoshop, click the "Channels" tab, highlight the red channel, select all (Ctrl + A), cut (Ctrl + X), and paste (Ctrl + V) the image into the green channel.   Fill the red channel with black. 
    6. When working in ImagePro, you can remove the numbers by clicking the "Options" tab under Measurements and then unclicking "display names on image."