Our protocol is significant because it uses the well-established technique of microinjection in Drosophila embryos to allow new research on the actin stress response and accompanying actin rod assembly. The main advantage of this technique is that we can study the actin stress response under a variety of contexts, such as a mutant screen or under different stress conditions. An individual who has never performed this technique before may struggle with handling the embryos and performing the microinjection.
We recommend practicing these steps until the user feels comfortable. Visual demonstration of this method is critical because the steps of embryo handling, positioning, and mounting for both microinjection and imaging might not be clear from written instructions alone. Prepare embryo collection cups and apple juice agar plates according to manuscript directions.
To promote the most generous egg laying, set up collection cups with flies two days prior to the experiment. Add at least 100 female and 50 male flies to each collection cup, then cover it with an apple juice plate. Prepare a working solution of G-actin red and store it on ice until it is loaded into the microneedle.
Allow flies to lay embryos for 30 minutes on the apple juice plates with yeast paste at 18 degrees Celsius. While the flies are laying, cut out a four by one centimeter rectangular wedge of apple juice agar with a razor blade and place it on a 25 by 75 millimeter glass slide. Harvest the plate from the collection and dechorionate the embryos by pouring fresh bleach solution onto the plate and swirling it for one minute.
Dampen the bristles of a paintbrush with distilled water, then use it to transfer dechorionated and washed embryos from the collection basket onto the prepared apple juice agar wedge on the glass slide. Use a pair of fine tip tweezers or a dissecting needle to arrange 10 embryos in a straight line along the long axis of the rectangular agar wedge. Arrange the embryos head to tail such that their dorsal side is facing the researcher.
Cut half a centimeter off the end of a P200 pipette tip with a razor blade and dip it into the embryo glue. Generously coat a five millimeter region along the long edge of a 24 by 50 millimeter rectangular coverslip and let it dry glue side up. Once the embryo glue has dried, gently place the coverslip glue side down on top of the row of aligned embryos on the agar, leaving two to three millimeters of space between the edge of the coverslip and the row of embryos.
Flip the coverslip over so that the embryos are facing up. They should be stuck in a line along one long edge of the coverslip with their ventral region facing the closest edge. Place the coverslip on top of 150 grams of fresh blue desiccant in a 16 ounce screw top jar and tightly screw on the lid.
Desiccate the embryos for 8 to 10 minutes, then remove the coverslip from the desiccant jar and tape each short side of the coverslip to a microscope slide embryo side up. Add two to three drops of hollow carbon 27 oil with a Pasteur pipette to cover the aligned embryos and protect them from further dehydration. While the embryos are desiccating, back load the previously prepared G-actin red supernatant into the microneedle with a microloader tip.
Attach the microneedle to the needle holder and tighten the screw. Place the slide with the mounted embryos onto the microscope stage and use the microneedle controls to bring the needle into the same focal plane as the embryos. Insert the microneedle into the embryo so that it hits the embryo in the middle of its ventral region at the embryo equator.
When the microneedle tip is visible inside the middle of the embryo, trigger the injection. After the injection, slowly remove the microneedle. Move the stage and perform the injection on the other embryos.
After all embryos have been injected, place the slide with the embryos in a humid incubation chamber and close the lid. Remove the slide with the injected embryos from the humid incubation chamber. Working quickly, gently pry off the double-sided tape pieces that were used to adhere the coverslip to the slide.
Stick two 2.5 centimeter long pieces of double-sided tape together and cut the tape in half lengthwise to make two strips. Stick 2/3 of the length of each tape strip onto the first coverslip, blanking each side of the embryos. Leave 1/3 of the tape strips hanging off the edge of the first coverslip where the embryos are stuck.
Gently place a second rectangular coverslip on top of the tape strips to sandwich the embryos between the coverslips, aligning the 25 millimeter edges but keeping the 50 millimeter edges offset from one another by one centimeter. Confirm that the heated stage is at the appropriate temperature. And if using an inverted microscope, add immersion liquid onto the selected objective lens.
Place the coverslip sandwich onto the stage carefully, making sure that the new imaging surface is the one touching the immersion liquid. Focus on an embryo that is in cellularization, then switch to the laser acquisition mode on the confocal microscope and adjust laser power and gain, frame size, tiling, and projection settings as desired. Take surface view images through the focal planes of the embryo's nuclei to find intranuclear actin rods.
Rods should appear in multiple orientations as bright streaks or dots inside the comparatively dark nuclei. The actin stress response and heat-stressed embryos is evident by the assembly of intranuclear actin rods. Actin rods appear in different orientations inside the nuclei and can be imaged through several focal planes.
In comparison, control embryos incubated at 18 degrees Celsius do not display actin rods. The percent nuclei containing rods can be quantified and FRAP experiments can be performed on the rods. An example of a fluorescence recovery plot for a bleached versus unbleached region of an actin rod is shown here.
When attempting this procedure, it is important to remember the actin rod formation is temperature dependent. For the steps following the microinjection, it is crucial to keep embryos at the desired incubation temperature. After microinjection, the researcher can perform FRAP to confirm the lack of F-actin turnover in the actin rods or to quantify changes in F-actin turnover occurring in other actin-rich structures.
