This method can help answer key questions in the developmental biology field, such as the mechanisms of neuronal cell migration. The main advantage of this technique is that you can detect individual and collective cell migration in the long term. After isolating 200 milliliters of bacterial culture by the alkaline lysis method, mix the two plasmids, such that the final concentration of each is four micrograms per microliter.
Incubate the fertile chicken eggs horizontally at 38 degrees Celsius in 70%relative humidity. After two and a half days, use a 20 milliliter syringe with an 18 gauge needle to eliminate five milliliters of albumin from the pointed side of the egg. Seal the hole with tape.
Next, use curved scissors to cut the top of the eggshell and open a hole, two centimeters in diameter. Under a stereomicroscope, check the developmental stage of the embryo. Seal this larger hole with tape.
Continue the incubation at 38 degrees Celsius and 70 percent relative humidity until E5.5. Before electroporation, prepare 10 microliters of the mentioned plasmid DNA colored with 0.5 microliters of fast green. Also, prepare 10 milliliters of autoclaved PBS containing 100 units per milliliter of penicillin, and 100 micrograms per milliliter of streptomycin.
Use a micropipette processor to prepare micropipettes made from glass capillary tubes. Cut the distal tip of the micropipette to an appropriate pore size for the amount of DNA to be injected. Use scissors to cut the sealed top shell to reopen a hole, two and a half centimeters in diameter, and set the egg stabily under the stereo microscope.
Deposit a few drops of the prepared PBS solution into the egg. Using two fine forceps, peel off the allantoic membrane covering the embryo, being careful not to cause a hemorrhage. Then remove the amnion from the head of the embryo.
While turning the head with a microspatula, use a micropipette connected to a suction tube, to inject between 0.1 and one microliter of the colored DNA into the ventricular cavity of the left optic tectum. Place a pair of forceps type electrodes around the target area of the optic tectum. Use the pulse generator to charge a pre pulse of 30 volts for one millisecond with a five millisecond interval, and for subsequent pulses of six volts for five milliseconds with a 10 millisecond interval.
After all this, seal the hole with tape, and continue incubating at 38 degrees Celsius and 70 percent relative humidity. Soak the electrodes in a plastic dish filled with PBS, and use an interdental brush to remove the albumen. Repeat the process from the initial shell cutting to the electroporation for each egg.
One day before the Flat-Mount Culture, began preparing the coded cell culture Insert by adding autoclave to distilled water to a 10 centimeter cell culture dish. Float some cell culture Inserts on the water. At a solution containing eight micrograms per milliliter laminin, and 80 micrograms per milliliter Polly L, making sure to cover the Inserts.
Incubate overnight at 37 degrees Celsius in the cell culture carbon dioxide incubator. On the day of the culture at E7.0, removed the laminin and poly l-lysine solution from the Insert. Then, transfer the Insert to a glass-bottom dish filled with 1.1 milliliter of culture medium.
Transfer the dish to the cell culture carbon dioxide incubator at 37 degrees Celsius. Set a humid chamber unit on an inverted confocal microscope with a gas flow of 40%oxygen and 5%carbon dioxide, and a temperature of 38 degrees Celsius. To begin preparing a tissue sample on the Insert, use forceps to pinch the embryo head out into a six centimeter cell culture dish containing ice-cold HBSS.
Use two fine forceps to isolate the electroporated optic tectum. And then use a plastic dropper to transfer it into a concave glass dish based with black silicon and filled with ice-cold HBSS. Check the position of the labeling under fluorescent stereoscopic microscope.
Using a micro surgical knife, cut out the tectal tissue surrounding the labeling while noting the direction of the tissue in the tectum. After this, use a plastic dropper to transfer the labeled tissue to the Insert so that the pin side is attached to the Insert. Lay the tectal tissue in the desired direction and remove any excess HBSS.
Repeat the tissue preparation process for any other tissue samples intended for the same Insert. Then place the dish in the preformed chamber of the inverted confocal microscope. Using the inverted confocal microscope, check the fluorescent labeling and focus the microscopic field.
Start the laser confocal unit. Sample the confocal scan in order to adjust the direction and position of the tissue along the x and y axes in the field. Next, select the scanning size.
Decide the interval and total range of the confocal scan along the z-axis. Take an interval of five or 10 micrometers for the 100 micrometer range. Choose the time interval of the confocal imaging and the total imaging duration.
After setting the parameters of the confocal running program, begin imaging. Once imaging is complete, combined the confocal images at a different C axis to produce z-stack images with final focal adjustment at every time point. Then append the Z stack images at different time points to construct a time-lapse movie.
In this study, cell migration is detected in superficial layers through electroporation in Ovo, flat mount cell culture, and time-lapse confocal imaging. The tangentially migrating cells and the nuclei in the superficial layers of the optic tectum are seen here zero hours, nine hours, 18 hours, and 27 hours after the onset of recording. A time-lapse movie of 10 minute intervals over a 28 hour and 50 minute period, shows the mass movement of migrating cells and their nuclei.
The merged movie clearly shows the mass movement of the migrating cells. The directionality of the cell migration can be examined by focusing on the dispersing cells from the labeled center to all directions. The clear images of the nuclear movement, allows for the automatic tracking of the nuclear migration using a particle tracking plugin.
Temporal changes of the trajectories of the tangential migration can be visualized and prove that the dispersing migration is omnidirectional. Higher magnification images can be taken in five-minute intervals. To investigate individual cell behavior with the sequential morphological change of the leading process, trailing process, and nuclei.
Following this procedure, labeling of illuminating actions can be performed. In order to answer additional questions like the mechanisms of Aksum guidance.
