This assay enables the student to look specific molecules in many developmental process, such as cell differentiation and morphogenesis by implanting the beads into distinct embryo regions. This protocol can be applied to any experimental animal models, cell culture, organotypic model, including organoids. Furthermore, it's a helpful educational tool for teaching basic developmental biology to students.
Demonstrating the procedure will be Alexandra Gonzales-Gonzales, an undergraduate student from the laboratory. To begin, incubate the fertilized white Leghorn chicken eggs vertically with the pointy side down in a humidified incubator at 38 degrees Celsius and 70%relative humidity. Once they reach the 28th stage, according to Hamburger and Hamilton, remove the eggs from the incubator, swap them with 70%ethanol and allow them to air dry.
Then disinfect the working area, microscopes and instruments with 70%ethanol. Next, candle the eggs to identify blood vessels and locate the embryo. Discard eggs that do not have an embryo.
Using the end of non toothed forceps, open a window by tapping the blunt end of an egg, and remove a one square centimeter shell section. Then, transfer the egg into a carton or plastic holder and place it under the stereo microscope. Using fine surgical forceps, remove any small piece of egg shell that could contact the embryo and remove the air membrane by puncturing and pulling it out.
Next, using fine surgical forceps, tear the amniotic sack slightly, being careful not to damage the vasculature of the Chorioallantoic membrane. Stage the embryos in-ovo to determine whether they are in the desired stage. For Affi-gel heparin bead preparation, cut a square shaped parafilm section to fit into a 45 millimeter Petri dish.
After placing the parafilm across the bottom of the Petri dish, using the end of non toothed forceps, push each vertex and fix it to the bottom of the dish. Next, using a pipette or spatula, transfer the beads into a micro centrifuge tube and wash them twice with PBS by allowing them to settle and pipetting the supernatant. Then, using a micro pipette, transfer 40 to 50 beads to the center of the parafilm covered Petri dish.
Under a microscope, select 30 Affi-gel or heparin beads that have a 100 micrometer diameter. Size the beads using a third interdigit of a 28 stage embryo as a reference. The bead must be smaller than the interdigit.
After carefully removing the excess PBS surrounding the selected beads, soak them in two to five microliters of the treatment solution. Incubate the beads in the solution for 30 minutes at room temperature. During incubation, to prevent the beads from drying out, pipette a few drops of PBS or water around the beads to humidify the local atmosphere and cover the dish with parafilm to slow evaporation.
Pour AG1X2 bead preparation. Use the spatula to transfer the beads into a micro centrifuge tube and add 50 microliters of the treatment solution at the desired final concentration. After wrapping the tubes with foil to protect them from light, incubate the beads for 20 minutes at slow shaking.
After incubation, using a pipette, remove as much of the treatment solution as possible in stain with 0.2%phenol red dissolved in water for two minutes with mild agitation in a vortex. After staining, remove the dye, then wash the beads twice in PBS to remove any residual dye. Next, using a micro pipette tip, transfer 40 to 50 AG1X2 beads to the center of the paraform covered Petri dish and soak the beads in five microliters of PBS.
Then under a microscope, select around 30 beads of 100 micrometer diameter in size. Submerge the selected beads in the experimental solution with a few drops of PBS or water around the beads to humidify the local atmosphere. Cover the dish with parafilm to slow evaporation.
Before manipulating the embryos, arrange two stereo microscopes next to each other on a benchtop. Then, using non toothed forceps, create a window in the remaining eggs. Next, under the microscope tear the amniotic membrane near the right hind limb with fine surgical forceps.
Using fine forceps, hold the embryo by the amniotic membrane to expose the right hind limb. Then using a fine Tungsten needle, make a hole centered in the distalmost of the third interdigit of the hind limb. Without releasing the embryo and the exposed hind limb, take one treated bead from the other microscope using one of the tips of the forceps.
The forceps must be open for one bead to adhere to its tip. Transfer the bead into the chick embryo near the hind limb, positioning it on top of the interdigit hole. apply pressure on the bead by closing the forceps until it enters the hole.
Then release the embryo, seal the eggshell window with tape and return the eggs to the incubator until they have reached the required stage. To ensure assay efficacy, the bead must be placed consistently and precisely in the correct location. In this study, the soaked bead was placed in the distalmost of the third interdigit beneath the apical ectodermal ridge.
The embryos can be stained with ocean blue and alizarin red to evidence the formation of skeletal elements and evaluate the effects on cell differentiation. This assay is also well-suited for evaluating cell death with neutral red and gene regulation, buying C2 hybridization. The main advantage of this experimental tool is to control the time and location of the soaked beads.
Combining the core positioning to precise developmental timing provides enormous possibilities to study cell differentiation processes.
