The overall goal of this procedure is to dissect and mount zebrafish embryos for optimal microscopic visualization of the spinal cord. This is accomplished by first micros dissecting and removing the oak ball. The second step is to remove the oak debris and pipette the embryos onto a slide.
Next, the excess liquid is carefully removed with a Kim wipe, followed by addition of mounting medium. The final step is to orient the embryos on the slide with an embryo poker followed by application of the cover slip. Ultimately, fluorescence or bright field microscopy is used to show distribution of post mitotic cell bodies and the trajectory of developing axons.
This method can help answer key questions in the field of neurobiology, such as what are the mechanisms responsible for patterning, differentiation, and axon guidance. Though this method can provide insight into many aspects of nervous system development, it can also be applied to other tissues such as developing muscle. Generally, individuals new to this method will struggle because dissection under a microscope is required Prior to beginning the experiment.
Make an embryo poker by gluing a pipette tip to a past pipette and then gluing a fishing line to the pipette tip. To begin the embryo dissection, place up to 30 zebrafish embryos. Age 24 hours post fertilization into a 35 millimeter Petri dish filled with PBS.
Under a dissecting microscope, secure an embryo's head with a pair of forceps while pulling the oak away with an insect pin. Then use the embryo poker to move the embryos to a part of the Petri dish that does not have a lot of yolk debris. Next, aspirate the embryos into a glass pasture pipette pulling up as little liquid as possible.
Then gently pipette up to 21 embryos onto a slide. Carefully wick away excess liquid using a laboratory wipe while avoiding touching the embryos, and then add one drop of mounting medium to the embryos. Next, use the embryo poker to orient the embryos on their sides in two to three rows.
Now, add a dab of petroleum jelly to each corner of a cover slip. Turn the cover slip so that the side with the petroleum jelly is facing down and place the cover slip on top of the embryos. The petroleum jelly acts to prevent damage to the embryos from excessive compression.
Gently tap the cover slip on each corner until the mounting media is touching the cover slip if necessary, using a pipetter in the range of 20 to hundred microliters. Add more mounting media next to the cover slip and allow the added media to wick underneath the cover slip. Now, use a laboratory wipe to completely clean around the cover slip.
Do not apply pressure to the cover slip as this can damage the embryos when properly cleaned, the area is dry and has no petroleum jelly or mounting media on it. Finish by using nail polish to seal around the edges of the cover slip. After mounting the zebrafish embryos, several HEMI segments above the oak sac extension were visualized.
This figure shows NK X 6.1 expression in the ventral spinal cord of the zebrafish embryo. The NK X 6.1 mRNA is distributed in roughly the ventral half of the spinal cord at 24 hours post fertilization. Within the progenitor domain, progenitor cells are distinguishable from post mitotic cells due to the presence of the floor plate, which is found in the medial part of the spinal cord.
Viewed with DIC optics. Individual cells are clearly distinguishable and a defined boundary between expressing and non expressing cells is evident. Progenitor cell cycle exit is accompanied by changes in gene expression.
This image shows robo three expression in post mitotic neurons. The robo family of receptors are axon guidance receptors whose expression allows for accurate counts of post mitotic neurons. Note that the floor plate and progenitor zone are not evident in this more lateral focal plane.
Confocal microscopy was used to image znp one immunofluorescence, which labels motor axons as shown in this laterally mounted embryo. The motor neurons exit the spinal cord ventrally at 24 hours post fertilization to innervate the surrounding developing musculature. Once mastered, this technique can be done in 30 minutes if it is performed properly.
While attempting this procedure, it's important to remember to preserve the integrity of the tissues through careful microdissection. After watching this video, you should have a good understanding of how to use microdissection techniques to remove the yolk of zebrafish embryos for mounting and microscopic visualization.
