The overall goal of this procedure is to better visualize stained fixed zebra fish embryos at early developmental time points. This is accomplished by first staining the zebra fish embryo using home mount in C two hybridization. Next, an embryo is selected to flat mount and a central incision is made into the yolk With a pair of fine forceps, then the lash tools are used to gently scrape and remove the remaining yolk granules.
Finally, the embryo is mounted in glycerol on a slide to enable the appropriate imaging of the specimen. Ultimately, flat mounting allows better visualization of stained embryos from a dorsal view by removing the yolk and positioning the embryo in a two dimensional preparation. The main advantage of this technique over existing methods like imaging intact embryos at younger developmental stages is that flat mounting removes the yolk sack allowing the entire embryo to be viewed.
This method can help answer key questions in the developmental field, such as characterizing the domain of the renal pre gender field in an embryo. Generally, individuals new to this method struggle because this technique requires delicate handling of the embryo, and also because learning the appropriate amount of pressure needed to remove the yolk sack can take time. Demonstrating this procedure will be myself, Zoe, and Amanda, who are graduate students in the winger laboratory.
After collecting, fixing, and staining embryos, according to the text protocol, select an embryo for flat mounting with one X-P-B-S-T. Transfer the embryo into a plastic or glass petri dish. Then with the dish under a stereo microscope, use fine forceps to roll the embryo so that a lateral view is obtained with the head and tail ends visible.
Next, use fine forceps to hold the embryo steady and a second set to make an incision in the yolk at a position centrally located between the head and the tail of the embryo. Then with fine forceps, scoop out the yolk from the oak cell cavity. Use one X-P-B-S-T to rinse stray yolk from the embryo using one to two drops of one XPBS.
Transfer the embryo to a flat glass slide. Drag the embryo to the edge of the buffer so that it remains in a flat position against the glass slide. While anchoring the embryo, use a lash tool to gently scrape the ventral surface to remove yolk granules.
If the PBST solution becomes cluttered with excess yolk or begins to evaporate, add one to two fresh drops and drag the embryo into this fresh buffer. After the yolk has been satisfactorily removed, gently transfer the embryo back into a Petri dish of PBST for a final rinse. Then transfer the embryo into a Petri dish containing 100%glycerol and incubate for five minutes.
To mount the embryo on a glass slide, transfer it to a clean glass. Slide in one to two drops of 100%glycerol and position it with the ventral yolk side facing the glass slide. Using a lash tool, drag the embryo to the edge of the glycerol drop so that it remains in a flat position against the slide.
If the embryonic axis is curved, use the fine forceps to gently make small incisions in the remaining yolk to relieve tension so that it can be positioned flat. On the slide. Place small divots of modeling clay on the four corners of an 18 by 18 glass cover slip.
Slowly place the cover slip on the embryo, angling the cover slip in order to minimize air bubbles in the glycerol. Finally, add an additional drop of 100%glycerol to the side of the cover slip to fill the space between the glass and to eliminate drifting, use a stereo or compound microscope to image shown here. A combination of probes was used along with two color wish to label developing renal progenitors that give rise to the kidney and the embryos were flat mounted in early.
So mite stages, renal progenitors are demarcated in a U-shaped pattern by their expression of PAX two A transcripts surrounding the parial mesoderm that concomitantly expresses delta C shown in red. When delta C was co labeled with C crox 20, A rostral subdomain of the renal progenitors was revealed that expresses delta C analyzing expression of PAX two a, SLC four A four, SLC 12 A three, and S-M-Y-H-C one at the 14. So mite stage enables mapping of the entire renal progenitor domain with PAX two A, and revealed that a subset of cells in the rostral subdomain expressed SLC four A four.
That did not overlap with the coddle subdomain of renal progenitors that expressed SLC 12 A three. In this figure embryos with mutations in the Retin Retinaldehyde dehydrogenase two gene required for the biosynthesis of retinoic acid or embryos treated with DEAB and ALD H inhibitor developed with reduced or abrogated expression respectively of WT one A, demonstrating that two color wish with the flat mount preparation is valuable for the study of cellular domains during organogenesis. In zebrafish with genetic mutations or when chemically exposed to small molecules Once mastered, this technique can take 10 to 15 minutes if performed properly.
Following this procedure, other methods like imaging can be performed to answer additional questions like where the boundaries of the kidney segments are located. After Watching the video, you should have a good understanding of how to flat mount a stained zebrafish embryo in order to better visualize the desired structures by the removal of the yolk.
