The overall goal of the following experiment is to obtain coronal viewing images of intact oph embryos using confocal microscopy that can be used to precisely measure embryo size and quantify protein and RNA expression levels. This is achieved by using a mounting media of gelatinous consistency to encase embryos within it and position them upright. As a first step.
The embryos are aligned over a thin layer of glycerin jelly and then covered with a second layer of this media. Next, the mounting media containing the embryos is cut into blocks, which can be flipped vertically to hold. The embryos in position for imaging results are obtained that show cross-section images visualized with any sort of confocal microscope.
Hello, my name is Milu. I'm a graduate student at the Meso Lab in the biology department at Case Western Reserve University. Today we are going to demonstrate to a mounting protocol to image PHI embryos in an abroad position using a confocal microscope.
We developed this method during our developmental biology course lab here at Case in collaboration with Dr.The main advantage of this technique over existing methods like the Endon imaging technique developed by the Mindin Laboratory, is that it allows for simple preparation and visualization of fixed embryos or tissues of any size and shape. We first had the idea for this method when we needed to visualize cross sections of very small embryos of other gesso species, which would be very difficult to hand slice. This method can help answer questions in the developmental biology field by allowing precise quantifications of gene expression and protein levels along the dorsal ventral axis.
Typically, embryos are mounted longitudinally. While it's possible to get Zack reconstructions of cross-sections, there's often a problem of photobleaching of the fluorescent eyes light scattering, and it is very time consuming. Our technique minimizes these problems because cross-sectional images of embryos are visualized along a single confocal point.
Generally, individuals new to this method may experience some difficulties while cutting the glycerin jolly blocks near to tip of the embryos. Visual demonstration of this method is critical because it requires careful handling. We have a demonstration today made by our students, Catherine Chen and catty To prepare drosophila embryos for upright imaging.
First melt glycerin jelly in a 55 degree Celsius water bath for 20 to 30 minutes until it becomes a homogenous liquid swirl. But do not shake the bottle in order to avoid the formation of bubbles. Using a cut P 1000 pipette tip carefully pipette one milliliter of melted glycerin jelly onto the slide.
To create a thin even first layer free of air bubbles, let the jelly solidify for five minutes on a horizontal surface. Then place the slide in a minus 20 degree Celsius freezer for five minutes. Alternatively, cover the slide with plastic wrap and place it at four degrees Celsius for up to five days.
Once the first layer of jelly is solidified, bring the slide to the scope and proceed to embryo alignment. Pipet a small amount of stained embryos and 70%PBS glycerol on the side of the first layer of jelly. Under the scope, align the embryo side by side along three to four columns per slide.
Use a hypodermic needle to scoop them using the slanted side of the needle. As a spoon. Carefully orient the heads of all embryos in the same direction by sliding them horizontally on the jelly medium with the aid of the needle.
The final configuration of the aligned embryos will be similar to that described for DNA injection. Using a twisted piece of Kim wipe, remove excess PBS glycerol from embryos. Carefully pipette between 50 to 300 microliters of melted glycerin jelly over the embryos, depending on the number of embryos to make a second layer of jelly.
Finally, cool down the slide at minus 20 degrees Celsius for five to 10 minutes, or store it at four degrees Celsius overnight. To remove the glycerol embedded embryos from the slide for imaging under a dissecting scope, use a razor blade to cut through the jelly in close proximity to the anterior and posterior sides of the aligned embryos. Hold the razor blade at a 90 degree angle.
To make straight cuts, add 100 to 200 microliters of cold PBT next to the cuts. To carefully scrape off the jelly next to the embryos. Using a spatula, PBT helps release the block of embryos.
Transfer the block to a dry microscope slide for further cutting. First cut smaller blocks with five to seven embryos within it. Second trim excess of glycerin jelly that may interfere with imaging.
Finally, flip the embedded embryos to an upright position with the aid of a razor blade and a needle. To visualize drosophila embryos using an inverted confocal microscope, place the jelly block containing embryos onto a cover slip. Ensure that the site of the embryo with the least amount of jelly is in closer contact with the objective.
If using an upright microscope to image, make four stacks of supra glue. Number one, cover slips to be used as supports and place the embryo block in glycerol in between the supports. Bridge the stacks using a longer cover slip for confocal analysis.
Place the specimen on the microscope stage. Check the working distance of the objectives to be used to find out whether you can image all the way through the embryo or only partially set up the confocal configuration in the computer to start imaging as shown here. If the protocol is done correctly, cross-section images of embryos with intact morphology can be observed because the embryos are left intact.
This method can be used to take precise measurements of embryo sizes and gene expression levels across the dorsal ventral axis. Once mastered, this technique can be done in 30 to 45 minutes. It is important to remove excess glycerol before pouring on the second layer of glycerin jelly over the embryos.
Otherwise, the two layers may split and make the cutting step More difficult. Also, you need to check the working distance of the objectives you're using to make sure that you can image the entire embryo or only part of it because the working distance creates a limitation of how much you can image. Make sure to cut the jelly closely to the side of the embryo that you will be positioned towards the objective.
Otherwise, you'll be imaging only jelly instead of the embryos. After watching this video, we hope you'll have a good understanding how to take cross-sectional images of Joseph Embryos. Thank you for watching us and good luck with your experiments.
