のライブ解剖ショウジョウバエ胚：抗体染色によってミュータントコレクションをスクリーニングするための合理化手法

Here we see the procedure for collecting embryos. Transfer flies from vials into the tubes of a five barreled collection chamber. Then put a grape agri plate on top of the chamber and allow the flies to lay eggs for a few hours.

When the embryos are at the right stage, transfer them from the grape aate to double stick tape with a paintbrush. Now roll the embryos out of their kons and line them up on an agar block with their ventral sides. Up in the middle, we see a slide with a rectangle of wax and a piece of double stick tape At one end, invert the double stick tape over the block with the lined up embryos and touch it to the embryos to transfer them to the slide.

They will now be dorsal side up immediately. Add PBS to the area of the slide enclosed by the wax rectangle so as to cover the embryos. The top shows a side view of the slide and the bottom shows a top view.

Cut a slit along the viel membrane with a glass dissecting needle. Poke the embryos in the brain region, lift them up and move them into rows on the glass. Then dissect each embryo to make a filet.

At the end of the procedure, you will have a slide with rows of filet embryos. It is now ready to fix. Hi, I am Peter Lee, a poster in the Kaizens lab in the division of biology at Caltech.

Hi, I'm Ashley Wright, a Caltech graduate student in Kaizens Lab. I'm Kaizen, a professor at Caltech, and my research group works on the development of the nervous system in fruit flies. Today we'll show you a procedure for dissecting live opal embryos.

We've used these methods to search for orphan receptor ligands. We do this by screening collections of lines over expressing cell surface proteins by live staining of embryos with receptor fusion proteins. We also use this procedure in the lab to study embryonic phenotypes and conduct genetic screens.

I've conducted a deficiency screen for motor acts on guidance phenotypes. So let's get started. Peter's gonna show you how it's done.

To begin, use a spatula to place a spot of yeast paste on a grape agri egg collection plate corresponding to each of the barrels of an embryo collection chamber. Next, inject carbon dioxide gas into the fly vials to put the flies to sleep. Transfer a few hundred flies directly into the collection chamber.

Then cover the chamber with the egg collection plate and press down Using a 20 inch long strip of labeling tape strap the chamber and plate together starting from the middle of the agar plate. The tape will overlap on the egg collection plate. After the flies wake up, incubate at room temperature in the dark for two or three hours in a horizontal position following the incubation, tap the flies down and replace the collection plate.

If fewer than 100 embryos are present on each sector of the removed plate. Continue performing two hour collections until enough embryos have been collected. Collected embryos can be pooled according to the specifications in the accompanying written protocol.

To age the embryos, place a wet piece of 90 millimeter circle filter paper in the lid of a Petri dish and place the collection plate with the embryos upside down on it. Incubate the plate for the desired duration and at the desired temperature, prepare a slide for embryo staging and sorting by placing double-sided sticky tape on a slide. Then place a rectangular slab of grape agar on the S slide.

The egger should be big enough to hold five to 10 rows of 10 to 15 embryos. Once the embryos have reached the desired stage, remove the embryos from the incubator. Transfer the aged embryos from the collection plate to the double-sided sticky tape.

Using a wet paintbrush, spread the embryos evenly across the tape. Under A GFP dissecting microscope SOAR embryos for appropriate GFP expression, which indicates genotype. In this experiment, genotyping is performed by selecting embryos that lack expression of the balancer curly arm, GFP.

Since embryo genotyping will vary depending on the balancer used, please refer to the accompanying written protocol for more information about performing this.Step. Next, using a blunted dissecting needle coate the embryos by gently rolling them across the double-sided sticky tape. At stage 16, the gut has divided into three bands.

After this, the gut begins to develop diagonal bands near the tail and then coils and the embryo becomes clearer. Next, use the dissecting needle to roll each embryo on top of the corion or other embryos. Avoiding contact with the tape, lift the embryo and transfer it to the slab of grape agar.

Align the coated embryos on this slab with their dorsal sides against the agar slab and posterior ends facing down. Then place the slide in a Petri dish with wet filter paper and incubate the embryos at room temperature until they reach the desired age For dissection, prepare a slide for live dissection by cutting a small rectangular piece of double-sided sticky tape. Place the tape near one end of a super frost plus slide using a wax pen.

Draw a rectangle the width of the slide and 30 to 40 millimeters long around the tape. There should be approximately three millimeters of space between the wax and tape. Then transfer the embryos from the great plate slab onto the tape by inverting the slide and gently lowering it onto the lined up embryos so that the tape comes into contact with them.

The embryos should be oriented with posterior ends down. After the transfer, the embryos will be positioned dorsal side up on the slide immediately add one XPBS over the embryos to fill the wax rectangle using a glass dissection needle, which is made on a needle puller like an injection needle. Cut along the dorsal midline starting from the posterior end of the embryo.

Now poke the anterior end of the embryo and lift it up. Starting at the bottom, transfer the embryo to the right of the tape strip, keeping it in the buffer. Continue dissecting and transferring embryos, making an orderly row of embryos on the slide that matches each row on the agri slab.

Using the dissecting needle, gently push down the flaps of body wall onto the slide. Avoiding stretching or removing material from their surface. The needle should only contact the anterior and posterior corners at the dorsal edges of the body wall.

Since motor axons do not extend this far. Dorsally, avoid touching the needles of the slide as it will break after all the embryos have been transferred and affixed to the slide. Remove the gut from each embryo by severing the four gut from the brain area and pulling the entire gut away from the embryo.

Displace the hind gut out onto the slide. Once all dissections have been completed, use two past pipettes to simultaneously deliver room temperature, 5%formaldehyde to one corner of the wax, rectangle, and remove solution from the opposite corner. Exchange the fixative solution three times using a total volume of about six milliliters.

Incubate the embryos in the fixative solution for 45 minutes. It is very important not to allow the meniscus to contact the embryos as this will destroy them. Use a past your pipette to remove most of the fixative solution.

Wash with PB S3 times using the two pasture pipette method. Then wash with PBT three times to perme the embryos. This step also renders the embryos less sensitive to the meniscus.

After the final wash, carefully remove the PBT by tipping the slide and touch a Kim wipe briefly to the corner of the area enclosed by the wax to remove excess solution. Using a preloaded pipet man immediately add 200 microliters of blocking solution block for 10 to 30 minutes. At room temperature, remove the blocking solution using a Kim wipe wipe.

Then immediately add 200 microliters of the desired primary antibody diluted in blocking solution. Place the slide in a humidified plate and incubate overnight at four degrees Celsius. In this experiment, the monoclonal antibody 1D four is used to label motor axons.

The next day, remove the dishes from four degrees Celsius and wash the embryos three times on a rocking platform with PBT for at least 15 minutes per wash. For all wash steps, ensure that there is sufficient PBT to completely immerse the slide during the rocking. After the embryos have been washed, block the slide as before.

After blocking immediately add 200 microliters of secondary antibody and blocking solution and incubate two hours at room temperature here an Alexa 4 88 go anti-US. Secondary is used in conjunction with Rodine Phin to label muscle fibers. Again, wash three times with PBT on the rocking platform for at least 15 minutes per wash.

Transfer the slides to a different tray to remove residual detergent. Then wash two times with PBS for five minutes. Remove the slide from the PBS plot most of the remaining PBS from the well with a chem wipe, and immediately add 500 microliters of 70%glycerol.

Since glycerol has the same refractive index as the embryo performing this step will make the staining more visible, clear the embryos for two hours at room temperature or overnight at four degrees Celsius. Then remove the glycerol with a kim wipe. Removal of nearly all the glycerol will flatten the embryos while removing less glycerol will result in a more three dimensional structure.

Once the glycerol has been removed, put on a number one cover slip. The embryos are ready for imaging dissected and stained embryos can be visualized by immunofluorescence microscopy. Here a dissected embryo has been stained with the motor axon labeling antibody 1D four.

This image is similar in appearance to the more commonly seen images in which HRP immunohistochemistry has been used for visualization using immunofluorescence. It is possible to obtain higher quality images in which the details of the motor axon branches are more clearly seen. Cos staining with rho domine labeled phin, which binds actin, allows for simultaneous visualization of the muscles.

This gives a clearer picture of the muscle layering and organization then can be obtained with HRP staining Here, 1D four or motor axons are shown in green Fein or muscle is shown in red. We've just demonstrated how to dissect live drosophila embryos from collections of mutant lines. When you're doing these procedures, it's important to start out with easy lines like lio, armadillo, GFP, balanced stocks, and just do a few lines at a time.

As you become more expert and faster with the dissections, you can do up to 10 lines per day and this will allow you to screen a collection of 200 or so mutant lines in a period of 20 days or so. So this is really a very efficient procedure once you become expert at executing all of the steps. So that's it, and thanks for watching and good luck with your experiments.