1Whitehead Institute for Biochemical Research, 2MIT - Massachusetts Institute of Technology
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Gutzman, J. H., Sive, H. Zebrafish Brain Ventricle Injection. J. Vis. Exp. (26), e1218, doi:10.3791/1218 (2009).
Part 1: Preparing for microinjection
Part 2: Preparing the embryos
Part 3: Injecting the brain ventricle
Part 4: Imaging
Part 5: Image Processing
Proper ventricle injection images are shown in Figure 1 panels A-D, while an example of incorrect injection images are shown in E-H. Proper injections have sharp edges and non-diffuse dye. Incorrect injection, which occurs when the needle is inserted too far into the ventricle and hits the brain tissue below, can result in dye that is visible outside of the ventricle space and in the yolk.
Figure 1. Ventricle injections of 25 hpf wild type embryos. (A-D) Correct injection method and (E-H) incorrect injection method are shown as examples. (A,E) Dorsal brightfield images with corresponding fluorescent images (B,F) and the overlay of the fluorescent and brightfield images (C,G) for both correct and incorrect injection methods shown. Arrows indicate regions where dye has gone beyond the brain ventricle space due to an incorrect ventricle injection (see “Troubleshooting”). Anterior is to the left in all images.
|You are squashing the embryo with the needle.||Your needle is too blunt or the tip is too large.||Start with a new needle and do not break it as far back. Bevel your needle to make it sharp like a syringe needle.|
|The dye is throughout the embryo and the yolk (see Fig 1E-H).||Your needle went through the tissue and didn’t stay in the ventricle space.||Your needle may not be sharp enough, so you are unable to control the speed of puncture.|
|The dye is too faint or not in the forebrain.||You did not use a high enough pressure for the injection, or your needle was not far enough anterior to reach the forebrain when you injected the dye.||Increase your injection pressure or place your needle slightly more anterior during the injection. You can put the needle through the midbrain-hindbrain boundary if more detail is required in the forebrain. You can also simply wait a bit for the dye to further diffuse before imaging.|
|Your fluorescent images are dark around the edges after the color replacement in Photoshop.||You forgot to change the “fuzziness” setting in the color replace window.||Move the “fuzziness” all the way to the right in the color replace window.|
|The needle will not puncture the hindbrain roofplate.||Your needle is not cut properly.||Make a new needle. If you are doing a lot of injections you may need several needles as they can get dull.|
In this video we demonstrate how to inject fluorescent dye (Texas-Red Dextran) into developing zebrafish brain ventricles. This method is used to visualize the brain ventricle space in contrast to the surrounding neuroepithelium and is extremely useful for determining the shape of the ventricle space as well as the shape of the surrounding brain tissue. It allows us to better understand the process of brain ventricle formation and brain morphogenesis over time in the live embryo. This technique is an excellent tool for studying brain ventricle formation defects and for initial characterization and study of brain morphogenesis mutants.
The authors would like to thank Laura Anne Lowery who developed this technique in the lab. HS is supported by NIHMH70926. Jennifer Gutzman was supported by the CSBi/Merck postdoctoral fellowship.
|Forceps||Tool||Fine Science Tools||11252-20|
|Capillary Tubes (needles)||Tools||Frederick Haer and Co.||30-30-1|
|Agarose-Coated dishes||Tools||Agarose (Seakem GTG; Lonza) Dishes (Corning)||Agarose (50027) Dishes (430166)||Make agarose approximately 2-3 mm deep in the dish depending on the stage of embryo you are injecting|
|Dissecting Microscope||Microscope||Carl Zeiss, Inc.|
|Microscope Camera with Fluorescence capabilities and with image capture software||Tools|
|1-200 μ pipette tips||Tools||USA Scientific, Inc.||1111-0806||These are the perfect size to poke the holes in the agarose and have the embryos fit perfectly.|
|Embryo Loop||Tools||Make these yourself with a glass pipette and hair or fishing line and seal with beeswax.|
|Needle Puller||Tools||Sutter Instrument Co.||Settings (Heat 785, Pull 70, Velocity 40, Time 70).|
|Fluorescent dye Texas-Red Dextran 70,000MW||Reagent||Molecular Probes, Life Technologies||D1830||Other Molecular weights are available if desired.|
|Tricaine (3-aminobenzoic acid ethyl ester)||Reagent||Sigma-Aldrich||A-5040|
|Embryo Media||Reagent||According to Westerfield (5)|