In Utero electroporation of MAGIC Marker is a powerful and versatile technique for quickly individualizing and assessing the morphology of cortical astrocyte. The main advantage of this particular technique is to label multiple progenitor cells and the neuronal and viral progeny with a precise control. Demonstrating the procedure with me will be Karine Loulier, the principal investigator from Before beginning the procedure, sterilize all of the surgical tools at a high temperature in a glass bead sterilizer.
And place a drop of electrode gel into a 35 millimeter dish. Insert a micropipette into a micro injection holder and break the tip of the micropipette. Aspirate the DNA solution and place a one microliter drop a fast green solution with a pipette into the lid of a three centimeter dish as reference.
Then add a volume from the broken micropipette next to the reference drop and readjust the pressure or time parameter if necessary;so that the equivalent size drops will be produced by the micro injector. If the volume is smaller than the reference one, break the tip of the micropipette again and repeat. To prepare the pregnant mouse, after weighing and anesthetic injection, confirm a lack of response in the anesthetized female mouse and apply ointment to the animal's eyes.
Gently shaved the abdomen of the mouse placed in the supine position on a warming pad. Wipe the exposed skin with one pad soaked with iodine and one pad soaked with alcohol. And place sterile compresses around the shaved, cleaned and sanitized surgical area of the animal.
When the mouse has been prepped make a two centimeter vertical midline incision in the lower abdomen. And gently manipulate the embryonic bags to expose the uterine horns. Assess the location of the cervix and the number of embryonic bags on each side of the cervix.
And orient the brain of the embryonic day 15.5 embryo to be electroporated so that the brahma, which is easily recognized at the junction of the three main blood vessels that run along the cerebral fissures, can be visualized. Imagining a virtual line between the brahma and the eye, introduce the micropipette between the virtual line and the longitudinal fissure. Then press the foot pedal to deliver one microliter of DNA solution into the lateral ventricle of the targeted hemisphere.
A successfully injected ventricle will appear blue. After the DNA has been injected apply electrodes on both sides of the embryo with the anode covering the injected hemisphere. Press the foot pedal of the electroporator to deliver a series of four 50 millisecond 35 volt charges, separated by 950 millisecond intervals.
Then hydrate the embryo with warm saline solution. When all of the targeted embryos have been electroporated return the uterine horns to the abdomen and fill the abdominal cavity with warm saline. Close the abdominal muscle with a continuous absorbable suture.
And close the skin with approximately 10 individual stitches of a 4-0 absorbable suture. At the end of the surgical procedure, place the skin sutures regularly along the incision. Then place the animal in a clean cage with monitoring until full recovery.
The fully recovered pregnant mouse should have a neat hair coat and an intact surgical incision. For multi-channel confocal imaging of the injected tissues, set the microscope for excitation of the appropriate lasers. And locate the brightest astrocytes in the electroporated area.
Because the labeled cells closest to the surface may appear brighter than those deeper within the slice, adjust the acquisition settings on the surface cells to avoid saturating the images, using the high-low lookup table. An adequately balanced image should display only a few blue and almost no red pixels. Then use the motorized stage of the microscope to acquire tiled 1024-by-1024 pixels Z stacks with a 10%overlap between adjacent stacks to subsequently enable mosaic reconstructions of the electroporated area or the zone of interest.
The labeled astrocytes can be assessed with tiled confocal images acquired with a 20x, 0.8na objective and assembled as Z stack reconstructions as demonstrated. The contour of individual astrocytes can be delineated on each individual optical section of the confocal image stacks for segmentation and reconstruction of the territorial domain occupied by each astrocyte. From the same Z image stacks, the branch morphology of singled out cortical astrocytes can be segmented using open access software that allows extraction of the skeleton of the main astrocyte processes.
Proper hydration during the surgery and before the closing suture are placed is critical to boost the survivor of the embryos and the recovery of the mother. In Utero electroporation of MAGIC Marker toolbox can be used to label progenitor cells in distinct model at value stages of development to trace lineage at the single cell resolution.
