の透過方法

The overall goal of the following experiment is to prepare viable drosophila embryos with a permeable eggshell to test the bioactivity of introduced small molecule drugs or toxins. This is accomplished by first treating developmentally staged embryos with dilute bleach to remove the outer chorionic eggshell layers. The second step is to perme the embryos with a solvent that depletes the thin waxy eggshell layer that blocks small molecule solutes.

The third step is to treat embryos with a drug or toxin and a dye to simultaneously gauge the permeability of the eggshell and introduce a compound of interest. After further development, the embryos can be either directly imaged or processed for immunohistochemistry. Ultimately, the results can show the activity of a drug or toxin on the development of tissues as determined by fluorescent microscopy with immuno reagents or vital ties.

Today I'll be presenting our method of permeation of drosophila embryos for assays of small molecule activity. The main advantages of this technique over existing methods like the use of conventional organic solvents such as heptane to perme. The eggshell is that our embryo permeation solvent, or EPS is water missable and relatively non-toxic.

These properties facilitate embryo treatments whereby solvent exposures can be more precisely controlled and can be carried out in physiologically relevant media, both of which result in greater embryo viability. I first had the idea for this method when I contemplated how to remove the waxy layer of the egg shell. Being a skier, I reflected on the common use of citrus solvent to remove the residual wax from the base of skis.

This prompted me to translate this application to the fly embryo eggshell, and after some trial and error led to the resolution of the EPS composition, Prepare population cages with at least 500 mating age flies of the desired strains. The cages should be fit with 10 centimeter grape juice. Aerates with a dab of yeast paste.

Change these plates twice a day. After a few days, the flies will be conditioned to the culture and lay embryos more regularly. EPS is made out of surfactants and limiting solvent solutions and is good for two months.

When stored at room temperature, warm the surfactants to 37 degrees Celsius when mixing up fresh EPS For EPS treatment embryos are handled with baskets made from three centimeter, so 50 milliliter polypropylene tube and nylon mesh roughed up. Plastic edges melt to the mesh more easily during later development steps. Embryos are also handled with baskets made from the caps of 50 milliliter tubes.

The cap is drilled out and notched, and the threading between the cap and tube secures the mesh. Alternatively, a locally machine slide chamber is used and prepared with a piece of oxygen permeable. Do membrane secured over the opening by a retainer ring to harvest the embryos.

Set a fresh grape plate with yeast paste on the culture in the morning. After an hour, change the plate and discard the first collected plate. Let the second plate collect embryos for two hours at 25 degrees Celsius.

Then collect the plate and transfer it to 18 degrees Celsius.There. Allow the embryos to develop to the desired stage. When the embryos are ready, dec coate them Prior to the EPS treatment.

Rinse them off the plate into the mash basket with water and the use of a paintbrush. Second, rinse them in gentle running water to remove any debris. Third, immerse them in 50%bleach for two minutes with periodic circulation of the bleach solution.

Then promptly rinse them off with running water. Now fill six 60 millimeter dishes each with 10 milliliters of PBS to function as baths and make a one to 40 dilution of EPS stock in MBIM swirl the mix. A white emulsion will form blot the excess water from the bottom of the mesh basket using a wipe and immerse the embryos into the EPS dilution with constant swirling motion.

After swirling the embryos in EPS for 30 seconds, remove them and blot away the excess solution. Then move the basket between the six PBS baths, gently squirting to rinse and proceed with the dye and drug treatment. Optimizing permeability and viability is the single most tricky part of this protocol.

Permeability is sensitive to the embryo developmental stage. The temperature at which the embryos are aged and can also vary widely between individual strains of flies. The best parameters for EPS dilution and time of exposure must be arrived at empirically.

First, prepare a 50 micromolar solution of sci-fi carboxylic acid dye in M-B-I-M-T one milliliter in a microfiche tube is enough. A toxin or drug can be added at this stage for acute treatments vortex to mix. Transfer the perme embryos from the basket to the micro fuge tube with a paintbrush.

Cap the tube and invert it several times. Then load the tube onto a mutator and let them incubate for 15 minutes. After the embryos have settled, remove the dye with a pipette and add back one milliliter of M-B-I-M-T for a wash.Resus.

Suspend the embryos, let them settle and replace the M-B-I-M-T. Then do this for a third and a fourth time. Once the fourth washe is complete, remove all the M-B-I-M-T and move the embryos to a development basket for long development periods.

For the development basket first, prepare the basket with a rinse of 70%ethanol and then di water, blood dry with a lab wipe. When done. Next, transfer the basket to a 60 millimeter dish with six milliliters of incubation medium containing the desired drug or toxin.

Transfer the embryos to the mesh in the basket with a paint brush by gently dropping incubation medium over them, disperse the embryos into a monolayer. Be careful not to trap air bubbles under the mesh. To use a slide chamber first, prepare the chamber, invert the slide chamber and put a thin bead of vacuum grease around the edge of the opening.

Next load, 150 microliters of the desired medium onto the surface of the do membrane within the opening. Now, transfer the embryos to the media using a paintbrush and disperse them over the membrane. Finish by applying a 25 millimeter circular cover slip flattening the medium.

Apply a light pressure around the perimeter to ensure the seal. There is a robust effect of rearing embryos at 18 degrees Celsius on their ability to be perme by EPS and take up dye at late stages of development. This is illustrated by the absence of Rumine BD uptake in EPS treated embryos raised at 25 degrees Celsius sci-fi carboxylic acid dye uptake seen here in red reveals the various levels of permeability typically seen in EPS treated embryos.

Dynamic distribution of the far red sci-fi dye during development, and its consolidation in the yolk within the developed gut seen with blue autofluorescence reveal a criterion used to assess viability. The sci-fi dye can also determine prior embryo permeable subsequent to formaldehyde fixation and immuno staining. After EPS embryos were treated with sci-fi dye and methyl mercury toxin, they are stained with Antifa two seen in green to label motor neurons and anti E lab in red to label neuron cell bodies.

Sci-fi dye is pseudo colored in blue. The methyl mercury has an effect on the patterning of the cortone neuron cell bodies marked by the arrows in the control treatment embryos. These neurons have a more orderly fascially two shown here in white illustrates the normal branching of segmental neurons in the control treatment noted by the arrowheads.

In comparison, methyl mercury treatment changes the branching pattern as marked by the solid green arrows. The hollow green arrow marks where branching was displaced. Once mastered embryo permeation treatments in this technique can be done in about 30 or 40 minutes if it's performed properly.

Following this procedure, other methods like fixation and immuno staining of the embryos can be performed in order to answer additional questions such as what specific tissues are targeted by a drug or a toxin during development. I thank you for watching this procedure and I wish you the best of luck putting these methods to work in your laboratory.