( Microprojectile Preparation ...)Biolistic Transfection of Organotypic Brain Slices

Microprojectile Preparation
The protocol for coating 1.6 m gold particles and loading macrocarriers is the same as described above for transfecting cell lines, except that for slice transfection, we never use more than one standard recipe of gold for 6 shots. Standard recipe = 50 l of a 60 mg gold/ 1,000 l distilled H₂O stock solution.

Transfecting Brain Slices
We have made several modifications to the procedure recommended by Bio-Rad which are crucial to obtaining healthy, transfected neurons in brain slices, although these changes are not important for transfection of cell lines. The primary issue in determining slice health is balancing a sufficiently high helium pressure for gold particle penetration of the slice with the need to minimize the tissue-damaging shock wave resulting from high helium pressures. For slices, we have found 1,100 psi of helium to be optimal. Three modifications have been applied to decrease the damaging effects of the rupture shock wave. First, a 3" square piece of nylon mesh (95 m mesh opening, 39% open area; Small Parts, Miami Lakes, FL) is cut and attached to the base of the launch assembly, serving to baffle much of the helium shock wave. Second, the Millicell insert containing the slices is placed onto an inch square slab of agarose (1.85%, 0.25" thick) on a cooled aluminum block (approx. 2"x 2"x 0.5") on the target tissue shelf 9 cm away from the launch assembly. The agar acts to absorb the residual shock wave while cooling ameliorates secondary neuronal trauma. Finally, the chamber is flushed with helium through a custom-fitted access valve prior to pulling the final vaccuum so that the residual gas is helium and not
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