Culture Slice organotipica del mesencefalo ventrale embrionali: un sistema per studiare dopaminergici sviluppo neuronale In vitro</em

The overall goal of this procedure is to generate organotypic slices from the E 12.5 neuron embryonic midbrain for the analysis of developing dopaminergic neurons. This is accomplished by first dissecting the embryonic brain. Then the brain is embedded into low melting aeros.

The third step is to section the brain slices using a vibrator. The final step is to choose the right brain slice for culturing and time-lapse imaging. Ultimately, time-lapse imaging and immunofluorescence microscopy are used to show the early steps of dopaminergic neurons, migratory behavior and differentiation.

This method can help to answer key questions in the neurodevelopmental field, such as following neuron and migratory behavioral during the developmental ventral brain. One day before the dissection, prepare one X Krebs buffer and the culture medium according to the accompanied manuscript about two hours before dissection. Prepare 100 milliliters of 4%low melting aros in Krebs buffer by microwaving the solution until the aros is completely dissolved.

Then place the aros in a 45 degree Celsius water bath. Next, fill up the Vibram buffer tray with ice cold Krebs buffer. Turn the cooling element on and keep it at four degrees Celsius.

After that, fix a razor blade in the blade carrier. Prepare a scalpel, a fine paintbrush, and a mini perforated spoon for picking up the slices in the vibrator area for later sectioning. Then add Krebs buffer to the sterile Petri dishes and keep them on ice.

In the dissection area, prepare three large sterile Petri dishes for dissection and two small sterile Petri dishes for embedding a pair of small scissors, two pairs of fine forceps, a mini perforated spoon, a glass pasture pipet fire polished with a round closed tip and one liter of Krebs buffer on ice. Wipe all the dissection tools with 70%ethanol. Next, add culture medium to the wells of a six Well plate and incubate it at 37 degrees Celsius for two hours.

Next, add 1.5 milliliters of Krebs buffer with penicillin streptomycin to each well of another six well plate under sterile conditions. Place milli cell membrane inserts in the wells. Place the six well plate next to the vibrato so that the brain slices can be transferred onto the filter membranes.

Immediately after sectioning, sacrifice a pregnant female mouse with embryos at stage E 12.5 and remove the uterus by lifting it up with a pair of forceps. Then use another pair of forceps to tear the myometrium away from the uterus. After that, place the uterus in ice cold Krebs buffer under the microscope.

Use a pair of fine forceps to separate the muscular wall of the uterus, riker’s membrane, and the visceral yolk sack from the embryo. Then remove the embryos from the uterus and move the embryo to a separate Petri dish with sterile Krebs buffer. Next, cut the head off each of the embryos.

Fix the head by piercing through it at the eye level with a pair of fine forceps. Use another pair of forceps to carefully remove the skin and the skull. Then carefully lift the brain up and transfer it into a Petri dish with sterile Krebs buffer.

After that, wash the brains in 4%Low melting point aros. Embed two to three brains at a time in 4%fresh, low melting point aros. Using a Pasteur pipette with fire polished round tip.

Then place the embedding dishes on ice as even as possible. Next, lift the brains until the bottom of the aros is solidified, so the brains can settle evenly and be parallel to the bottom of the aros block. When the aros has fully solidified trim the aros surrounding the brains.

After that glue, the aros block onto the specimen stage of the vibrator. When gluing the block, make sure that the ventral side of the brain is parallel to the platform. Section 300 micron thick horizontal slices at a frequency of 50 hertz blade amplitude of 1.1 millimeters and a speed of 25 millimeters per second.

Collect the brain slices by using the fine paintbrush to push the slice to a mini perforated spoon. Then transfer them to a dish with sterile ice cold Krebs buffer under the microscope. Choose the slice that contains ventral midbrain tissue.

At this step, use a mini perforated spoon and a fine paintbrush to transfer the brain slices onto milli cell membrane inserts in a six well plate with Krebs buffer that was prepared earlier. Next, transfer the membranes with the slices to another six. Well plate with culture medium, keep the top of the membranes dry so that the brain slices receive medium from below and air from above.

Place the slices in an incubator with 5%carbon dioxide at 37 degrees Celsius. It is very important that the slices are placed in the incubator within two hours after the initial step of the dissection. These slices can be maintained in vitro for three to four days.

After four to five hours of recovery. The slices can be used in time-lapse imaging right before imaging. Add 1.5 microliters, 200 millimolar ascorbic acid to the dish to protect the slices against phototoxicity.

Incubate the slices in an environmental chamber at 37 degrees Celsius with 5%carbon dioxide during time-lapse imaging. Here is an acute slice showing the normal location of dopaminergic neurons in the ventral midbrain in which the projections to the forebrain have not yet developed. After one day in culture, the projections to the forebrains start to form as indicated by the yellow arrow.

White arrowhead indicate the location of the cell bodies shown in higher magnification in the insets. Here is the migratory root of YFP labeled neurons. In an acute horizontal slice, the initial position of the cells is marked with a red arrowhead.

After watching this video, you should have a good understanding of how to prepare genotypic slices from the E 12.5 marine embryonic midbrain to obtain viable and intact slices. It is important to perform the dissection of the embryonic brain carefully and after vibrato sectioning to choose the correct brain slice for culturing and TimeLapse imaging.