The overall goal of this procedure is to investigate the properties of floor plate derived signals in a variety of developmental contexts based on the production of floor plate conditioned medium. This is accomplished by first isolating the spinal cord from the mouse embryo at E 12.5 and dissecting out the floor plate. The second step is to cultivate the floor plate in a plasma thrombin matrix to produce floor plate conditioned media.
Next, the spinal cords from mouse embryos at E 12.5 are isolated and dorsal tissue containing comm, choral neurons are dissected out. The final step is the tation and exposure of dissected comm tissue to samples of floor plate conditioned medium. Ultimately, the tissue is processed for biochemical analysis such as western blot to show the commissural markers expression.
This method can help zuki question in the field of developmental biology. Signals released by the floor plate play crucial roles regulating cell fat, differentiation, proliferation and axon guidance tissue and cells from various origin. ZA be exposed to floor plate condition medium, demonstrating the procedure will be kind to an A technician from the laboratory.
Begin this procedure by placing a euthanized gestational E 12.5 pregnant mouse ventral side up. Soak the abdomen with 70%ethanol. Next, pinch the skin of the abdomen with forceps and cut through the skin and the peritoneal layers with surgical scissors.
Make an incision to expose the abdominal cavity. A string of embryos is present on each side of the mouse. Grasp one horn of the uterus in the tissue between two embryos and dissect it out starting from the exterior part.
Then transfer the intact uterus to a 100 millimeter Petri dish with cold PBS on ice. Next, extract the embryos from the extra embryonic tissues. Grasp the dark red placenta tissue with two pairs of forceps and pull softly to tear the tissue and remove the embryos from the uterine sack.
In a dissection hood, place one embryo in a drop of cold HBSS glucose. After decapitating the embryo, place the embryo ventral side down with the anterior part facing the experimenter. Pinch the skin of the embryo with one pair of forceps and pull away the skin with the other pair.
Repeat this step until the skin is completely removed from the anterior part of the embryo. Then turn the anterior side of the embryo to the left with one forceps. Pinning down the embryo in the anterior part.
Have the other forceps grabbing the skin and pulling toward the rostral side to expose the spinal cord. Next, use one side of the forceps to cut the meninges that are still wrapping the spinal cord. The spinal cord is now open.
Then turn the embryos anterior your side to the right. Detach the tissue from the spinal cord, starting from the cervical side of the embryo by sliding the forceps under the spinal cord and detaching the surrounding tissue and dorsal root ganglia with small rotatory movements. Then place the isolated spinal cord in a fresh drop of cold HBSS in a flat position with meninges on the top and the anterior side away from the experimenter.
Subsequently, pin down the spinal cord via the remaining hind brain with one forceps and grab the meninges with the second forceps. Then peel off the meninges starting from the rostral side of the spinal cord with slow and constant movement for floor plate dissection. Pinch the hind brain with one forceps and cut the floor plate out with a scalpel.
Then conserve the dissected floor plate in neuro basal medium. Add room temperature. In this procedure, place several sterile cover slips in a 100 millimeter Petri dish.
Next pipette 20 microliters of plasma in the center of each cover slip. Then transfer two to four floor plates to each plasma drop. Add 20 microliters of HBSS thrombin to the plasma drop Carefully mix them with a pipette tip and avoid contact with the floor plate explants and keep the cover slips for a minimum of 10 minutes at room temperature to allow coagulation of the plasma.
The plasma clot retracts a few microns as a sign of the coagulation and the coagulation must not be stopped prematurely to avoid detachment of the plasma clot. Afterward, transfer each cover slip to a four well plate and add 500 microliters of neuro basal plus B 27. Incubate them for 48 hours at 37 degrees Celsius.
After 48 hours in a tissue culture hood, harvest the medium from each. Well carefully. Be careful not to pipette the polymerized plasma thrombin mix or any floor plate fragments.
Then store 100 microliter aliquots at minus 80 degrees Celsius. The commissural neurons are located in the most lateral part of the spinal cord. To dissect the commissural neurons, pinch the hind brain with one forceps and cut out the lateral part with a fine scalpel.
Then conserve the dissected tissue in neuro basal medium on ice. For immediate use to treat the comm choral neurons, lacerate the choral tissue with small scissors. Cut the tissue as small as possible to potentiate the accessibility of the cells.
Next, distribute the tissue evenly between the different experimental conditions. Control treatment and floor plate conditioned medium treatment. Subsequently centrifuge the tissue fragments at 800 times G for one minute at four degrees Celsius.
Then check the repartition between tubes if necessary. Redistribute and centrifuge again until fragments are equally distributed. After that, remove the supernatant, add warm floor plate, conditioned medium, or control treatments to the tissues.
Next, incubate the tissues at 37 degrees Celsius for 30 minutes and gently shake them every 10 to 15 minutes. Then centrifuge them at 800 times G for one minute and remove the treatment at the end. Add the lysis buffer.
In this experiment, dorsal spinal tissue were collected dilacerated and stimulated with the floor plate conditioned medium or control medium. The samples were lysed and processed for Western blot. The proteins were then probed with antibodies, plex and a one and beta actin.
Here the western blot illustrates an increase in plex and a one levels in the sample stimulated with the floor plate conditioned medium. The dot blot shows the expression of GDNF in floor plate conditioned medium compared to its control medium. Following this procedure or the method could be performed instead of being stimulated and processed for western blood.
The tissue containing the cells of interest could be dissociated, and isolated cells could be plated on cover slips. The floor plate condition medium can be applied on culture cells and the modification of cell behavior or protein expression could be assessed for example by immunochemistry. After watching this video, you will have a good understanding of how to de like different part of the spinal cord and how to stimulate acute fresh tissue before proceeding with biochemistry.
