Hi, I am Ham from the laboratory of Dr.Review Klar at the medical neurobiology department in the Hebrew University of Jerusalem. Hi, I'm Sophie Esman and I'm also from a Lars lab. Today we are going to show you a procedure for Innovo, electroporation and spinal cord open book preparation.
We use this procedure in our laboratory to study axonal trajectories of genetically defined neuronal population in the embryonic spinal cord. So let's get started. To begin this procedure, transfer fertilized white leghorn chicken eggs from room temperature to a humidified incubator, preferably with rocking trays at 37 to 38 degrees Celsius.
It is important to monitor the temperature and humidity of the incubator in order to obtain viable and synchronized embryos. The chick embryos are electroporated when they have reached hamburger and Hamilton or HH stage 18 to 20, which is an optimal stage for enhancer dependence specific expression. At this stage, the head lies at right angles to the trunk.
Also at HH stage 18 to 20, a vast set of extra embryonic blood vessels such as anterior, posterior right and left vital line vessels should also be seen. The chick embryos reach HH stage 19 to 20 after 66 hours of incubation, and at that time remove the eggs from the incubator After removing eggs, place them horizontally and wait for five to 10 minutes. The embryo is now located at the upper pole of the egg.
Prior to electro pering, the embryos prepare Hank's solution with 100 units per milliliter of penicillin and 0.1 milligrams per milliliter of streptomycin. Prepare a desired mixture of DNA at two to five micrograms per microliter and add a few grains of the fast green dye for easy visualization of the injected DNA. Have a mouth pipette, a sterile syringe needle, small scissors, and a tape ready as well.
Prepare micro capillaries by pulling 0.5 millimeter diameter glass capillaries with a pipette puller. Place the tungsten electrodes into the micro manipulators and connect the electrodes to the pulse generator. Set the voltage on the pulse generator to minus 30 volts the length of each pulse to 50 milliseconds and the number of pulses to three.
Proceed to electroporation before exposing and handling the embryos. Insert a syringe at the narrow pole of the egg and remove five to six milliliters of albumin from each egg. The albumin is removed to avoid it from spilling.
When cutting the in the next step, use the small scissors to cut. Open an oval window at the upper side of the egg after revealing the chick embryo, moisten it with 0.5 to one milliliters of hanks and penicillin streptomycin solution. Next, use a mouth perpet to load the glass micro capillary.
With a DNA dye mixture, a micro capillary of the correct diameter should load easily with DNA. Under a binocular position the embryo for injecting the DNA into the neural tube. Place the embryo with its tail toward you.
At this developmental stage, the spinal cord is clearly visible and therefore no contrasting techniques are required. The spinal cord is sealed at both the head and the tail end. However, with the micro capillary punctures more hole in the neural tube and penetrate it at a shallow angle, a micro capillary that is thin enough and of the correct diameter should penetrate the tube without damaging it.
Inject the DNA using a mouth perpet. A micro capillary of the correct diameter should release the DNA with a regular exhalation. The green dye should spread from the tip of the tail up to the vesicles of the developing brain.
Inject until the neural tube is filled with DNA electroporation is required for the injected DNA to enter the cells First. Place the electrodes in parallel to the neural tube and as close to it as possible without touching the tube itself. Make sure the electrodes are covered with liquid for allowing electrical conductivity during the pulse.
Usually the Hank solution added immediately after exposing the embryo is sufficient. But if not a one drop of Hank's solution just prior to the pulse with the electrodes positioned and submerged in liquid pulse to electro operate, then carefully remove the electrodes and seal the window and the egg with tape. It is important to seal the egg entirely to prevent drying of the embryo during the following incubation period.
Place the egg back in the incubator and incubate until the chick embryo reaches developmental embryonic. Day six or E six, when it is time for imaging when the electroporated embryo has reached developmental embryonic. Day six, E six three days after electroporation, remove it from the egg and place it into a silicone coated Petri dish containing PBS using fine scissors.
Cut away the membranes and stretch the embryo on its ventral side using pins pushed into the silicone to hold it in place. Using a sharp tungsten micro capillary. Make a longitudal incision along the roof plate from the hind brain down to the tail.
Make two additional longitudinal incisions on both sides of the spinal cord, detaching the dorsal root ganglia or DRGs from the spinal cord. Detach the floor plate from the tissue starting from the tail to the hind brain, leaving the spinal cord intact. Finally, use fine scissors to cut the spinal cord in a transverse section at the hind brain and separate the spinal cord from the body.
Now that the spinal cord has been isolated, spread it in a new silicon coated Petri dish containing PBS using pins to hold it from the hind brain to the tail producing a flat mount preparation. Fix the spinal cord in 4%paraldehyde in PBS for one hour at room temperature immuno stain following the immunohistochemistry procedure, detailed and accompanying written protocol to mount the spinal cord open. Book preparation for imaging.
Spread grease or silicone in a rectangle shape on a slide and drip PBS within the rectangle. Use tweezers to place the spinal cord, stretched the middle of the rectangle and draw out the liquid around it. With a pasta per pet place one to two drops of mounting media on the spinal cord and cover with a cover slip.
Apply pressure to the cover slip. To avoid air bubbles. Keep the slides in dark in four degrees Celsius.
Inspect the spinal cord using confocal microscopy. Here are some representative results of axonal rejections of chick spinal inter neurons in control experiments where RFP is expressed in all the neurons utilizing a ubiquitous enhancer element from CMV, most of the neurons and their axons are labeled. It is impossible to decipher the axonal traject of neuronal subpopulations.
However, when applying specific enhance elements, a defined axonal pathway of a specific subpopulation DI two neurons in this example is revealed. So we've just shown you how to electro operate a cheek neural tube with a specific neuronal enhancer at E three and how to prepare an open book spinal cord at E six. When you're doing this procedure, you should remember to be careful and patient.
So this is it. Thank you for watching and good luck with your experiments. Good luck.
