We aim to understand how mechanical and biochemical signals coordinate animal morphogenesis, including the detection and the response to forces by cells and molecules. More molecules have been discovered to be mechanosensitive, functioning differently under varying levels of tension. However, the physiological context and the function of these force-sensitive events remain largely unknown.
Due to the dynamic nature of force-sensitive events, our field largely relies on high speed fluorescence image and time-lapse recording to capture molecule and cellular behaviors. Many fluorescently tagged protein are not bright enough to be imaged, and it requires special temporal resolution without significant blanch. On the other hand, traditional immunofluorescence methods can only capture natural or molecule and cellular feature after fixation.
This method, the pathway to dynamical track of the localization of many no abundance protein receptors of major signaling pathways, such as Notch, WNT and the BMP pathways. Eventually, in addition to the traditional linear signaling cascades, we can pinpoint when and where the signaling events occur at the subcellular scales. To begin, add 110 microliters of the antibody mix to the tube containing Alexa Fluor 594 dye.
Invert the tube several times to mix the components thoroughly. Incubate the tube on a rotator for one hour at room temperature. Then assemble the purification column from the conjugation kit and prepare a 1.5 milliliter resin bed.
Centrifuge the column at 1100G for three minutes at room temperature to remove excess liquid from the resin. Add the reaction mix drop-wise to the top of the resin column. Centrifuge at 1100G for five minutes at four degrees Celsius to collect the label to antibody.
To begin place, 200 newly hatched adult drosophila in a plastic cylinder shaped cage. Seal one side of the cage with porous metal mesh for ventilation and the other with a fruit juice in yeast paste agar plate. On the day of injection, replace the cage's agar plate with a fruit juice plate containing minimal yeast paste.
Incubate the plate at 25 degrees Celsius for one hour to collect embryos. Then remove the plate from the cage to halt egg laying and incubate at 25 degrees Celsius for an additional two hours to obtain two to three hours old embryos. To remove the eggshell from embryos, introduced two milliliters of 50%bleach solution to the fruit juice plate.
Using a paintbrush, gently dislodge embryos from the plate and incubate the plate for two minutes while rotating it every 30 seconds to enhance the efficiency of eggshell removal. Then pour the embryo and bleach mixture into a 70 micrometer nylon cell strainer. Rinse the embryos comprehensively with a water bottle to eliminate any leftover bleach and yeast paste.
Using a clean razor blade, cut a rectangular agarose block and position the block on a glass slide. With a paint brush, transfer embryos from the strainer to the gel block and distribute them uniformly along the center line of the block by brushing gently. Now prepare a tweezer with its two legs securely taped together to form a single sharp point.
Under a dissecting microscope, pick individual embryos using the tweezer and align them along the gel blocks extended edge. Pipette 10 microliters of heptane glue at the midpoint of a 24 by 50 millimeter cover slip. Use a pipette tip to spread the glue into a 0.5 by three centimeter rectangular area.
Using a flat tip tweezer, lift the glue coated cover slip and hold it steadily above the embryos, ensuring the glue side faces the embryos at approximately 45 degrees. Gently press the cover slip against the gel block, allowing the embryos to touch the glue. Swiftly release the pressure and elevate the cover slip.
Then dispense 10 microliters of water at the center of a fresh glass slide. Place the embryo bearing cover slip on it, ensuring the embryo side faces up. Afterward, apply 40 microliters of halocarbon oil at one extremity of the embryo strip.
Angle the slide until the halocarbon oil covers the embryo's surface. To begin, fill each needle with five microliters of Alexa Fluor-labeled GFP Nanobody solution. Place a 25 by 25 millimeter cover slip on a glass slide surface.
Pipette 40 microliters of halocarbon oil on the slide, and spread it along the cover slip's perimeter. Under the injection microscope, position the needle's tip against the cover slips edge submerged in oil. Adjust the PicoPump's injection air pressure accordingly.
Replace the empty glass slide with the prepared drosophila embryo slide. Set the injection needle tip perpendicular to the embryo's anterior posterior axis. Use the XY stage manipulator to guide embryos toward the needle until the tip reaches the center of the yolk, and pump two to three times to infuse antibodies into the yolk.
With the XY stage manipulator, steer embryos away from the needle post-injection and move to the next embryo. Allow the embryos to incubate at 25 degrees Celsius until reaching the desired developmental stage. After incubation, identify the embryos under a low power lens with bright-field illumination, and switch to a 40x or 63x lens for high resolution fluorescent live imaging.
Post injection, the signal to noise ratio of the Notch receptor improves significantly comparable to the signal quality of immunofluorescence. Moreover, antibody injection allowed the temporal characterization of Notch localization at 45 second intervals without an apparent loss of signal intensity over a five minute imaging window. Live imaging of embryonic phosphotyrosine showed that tyrosine phosphorylation is highly enriched at the tricellular junctions.
Additionally, a novel second population of phosphotyrosine signal was observed underneath the center of the apical membrane. Dual color imaging revealed that this population of phosphotyrosine signal is near medial myosin. In addition, the medial population of phosphotyrosine exhibited similar pulsatile coalescence and dissipation patterns as shown for medial myosin.
