Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

The overall goal of this procedure is to label and modify single cells of the dentate gyrus and follow their development. This is accomplished by first injecting DNA into one hemisphere of the brain at embryonic day, 15.5 or 18.5. The second step is to electroporated the injected DNA into the developing dentate gyrus area.

Next brain sections are prepared by using a vibrator, and the brain slices are kept in culture for up to 14 days. The final step is the immunofluorescent staining of the brain slices using appropriate antibodies. Ultimately, confocal immunofluorescence microscopy is used to show changes of the modified granule cells of the developing dentate gyrus.

This method can help to answer key questions in neurobiology fields, such as role of specific genes in development of specific neuronal cells in different brain areas. In our case, BC 11 B transcription factor in development of gyrus. Begin the procedure by dissecting the uterus of a euthanized mouse, which contains the embryos.

Place it in a Petri dish containing 15 to 20 milliliters of cold complete HBSS on ice. Next, use a pair of scissors to separate each embryo from the uterine horn. Then place it in a second Petri dish containing cold complete HBSS under a dissecting microscope, sever the uterine muscle wall.

Carefully release the embryo from the yolk sack and the placenta. After that. Use a pair of bond scissors to decapitate the embryos just above the fore lims at a 60 degree angle.

If the experiment requires genotyping of the embryos, collect a tissue sample for genomic DNA isolation. Then transfer the head to a clean and dry Petri dish. Because the head has been decapitated in a 60 degree angle, it should tilt to one side when placed dorsal side up.

Now place a needle carefully into the middle of the hemisphere, close to the bgma. Inject approximately two to three microliters of DNA solution by stepping on the pedal of the pico spritzer. Three, using 30 pounds of pressure for the duration of 10 to 15 milliseconds per pulse.

Applying five to eight pulses with one second between pulses. Before placing the electrodes, apply a few drops of complete HBSS on the head of the embryo O.Then place the electrodes in such a way that the negative terminal is on the same side is the injected ventricle and the positive electrode on the opposite side of the injected ventricle below the ear of the embryo's head. Subsequently, apply five pulses of 50 volts.

After electroporation. Place the head of the embryo in a dish containing 15 to 20 milliliters of cold complete HBSS solution. Peel off the skin from the embryo's head.

Then make a small incision in the middle of the cerebellum at the midline of the skull. Insert the spring scissors into the incision and cut longitudinally along the sagittal suture. Peel off the skull and detach the brain from it.

Meanwhile, pour 4%LMP aros into a peel away mold and peel it at room temperature. Take the brain out of the complete HBSS by a small scoop spatula and drain the excess of HBSS with fine tissue paper or chem wipe. Place the whole brain gently into the agar roses and adjust its position with a fine needle.

Keep the mold on ice until the agar rose is solidified and the block is sectioned. Now trim the LMP agros block and glue it to the specimen stage using super glue. After the glue is dry, transfer the specimen stage to the buffer tray of the vibrator.

Fill it with ice cold complete HBSS until the block is immersed in the solution. To prepare for sectioning, set the vibrator to the appropriate settings. Cut the sections containing the desired tissue at slow speed, beginning from the hind brain, and collect five to seven sections from the cerebrum.

Then transfer the sections to a clean six well culture dish containing five milliliters of ice cold, complete HBSS and keep it on ice till all the sections are collected. Then place the membrane inserts into a six well plate containing 1.8 milliliters of slice culture medium. Moisten the membrane in a cross fashion with 100 microliters of complete HBSS before placing the sections on the membrane in order to facilitate the orientation of the sections.

Subsequently, transfer the sections onto the membrane. Place up to five sections on one membrane, and do not overlap the sections with each other. Take the excess of HBSS off the membrane using a pipette.

Incubate the culture dish at 37 degrees Celsius with 5%carbon dioxide for 11 or 14 days in vitro and change half of the medium every other day. At this stage, add reagents such as bromo deoxyuridine for labeling proliferating cells to the media for the first 20 hours of the culture time. In this step, use a clean and sharp scalpel blade to trim the membrane according to the orientation of the sections.

Transfer the sections along with the membrane to a 24 well plate containing one milliliter of 4%PFA and incubate the sections for one hour at room temperature. Next, wash them three times with one XPBS for 15 minutes each wash. Then incubate the sections overnight with permeable solution at four degrees Celsius with gentle agitation the following day, incubate the sections with the appropriate primary antibodies, diluted and permeable solution overnight or for 48 hours at four degrees Celsius with gentle agitation.

Afterward, wash the sections three times for 15 minutes with one XPBS and incubate overnight at four degrees Celsius with the appropriate secondary antibodies, diluted and permeable solution. After the incubation, wash the sections once with one XPBS for 15 minutes, followed by dappy staining for 10 minutes at the end, wash the sections three times with one XPBS for 15 minutes each and transfer to the microscope slides. Add immuno mount and gently.

Place a cover slip on top of the sections. Dry the slides overnight at four degrees celsius and seal with nail polish before analyzing them by confocal microscopy. Shown here is the injection of DNA expressing GFP into one hemisphere, followed by immuno staining at day 18.5 after electroporation.

This image shows the DPI staining GFP staining is shown in this image and the merged image is shown here. This figure shows the mosaic deletion of BCL 11 B by X utero, electroporation, followed by organotypic slice. Culture sections were immuno stained by using antibodies, recognizing GFP and NeuroD, the insets displaying GFP and BCL L 11 B.Staining at higher magnification demonstrate the loss of BCL 11 B expression in cells expressing Cree recombinase.

Here is the statistical analysis of GFP, neuro D and GFP neuro D positive cells. Once mastered, this technique can be done two and a half hours for four embryos if it is performed properly. While attempting this procedure, it is important to remember to handle tissue carefully and to avoid contamination.