Ex Vivo Electroporation of Chick Embryo Cerebellar Slices: A Method to Introduce Plasmid DNA Encoding Green Fluorescent Protein to Visualize Granular Cell Development

Construct an electroporation chamber by fixing the anode of an electroporator to the base of a 60-millimeter Petri dish using insulation tape. Add approximately 1 milliliter of HBSS to cover the electrode. Next, place a 0.4-micrometer culture insert on top of the electrode covered in HBSS.

Use a 3-milliliter Pasteur pipette with a cut tip to transfer the identified slices, up to 5 per insert onto the culture insert. Then, separate the slices and allow them to settle onto the culture insert in a sagittal orientation. Using a pipette, remove excess medium from the brain slices. Allow the insert to rest on the electrode so that there is contact between the insert and the electrode.

In this setup, the culture insert with the slices will rest upon the surface of the medium, maintaining the circuit but allowing spatial targeting of the cathode. Using a P10 pipette tip, pipette 5 microliters of DNA diluted with 20% Fast Green over the surface of a targeted region of each slice. The addition of Fast Green ensures that the DNA solution is viscous enough to prohibit wide dispersal of the DNA.

Then, place the cathode over the desired targeted tissue and electroporate the samples. Avoid direct contact of the cathode with the tissue by placing the cathode as close to the tissue as possible without actually touching it. Repeat the electroporation in multiple regions of the external granular layer on each individual cerebellar slice as desired.

When finished, transfer the culture insert to a 30-millimeter Petri dish. To each culture, add 1 milliliter of pre-warmed culture medium underneath the culture insert. Make sure that the insert does not float on the medium. The culture insert should be in contact with the medium, but the slices should not be bathed in it.