( or TrkC were co-transfected ...)Biolistic Transfection of Organotypic Brain Slices

or TrkC, were co-transfected with two expression constructs. One construct contained the rat cDNA for the TrkC receptor (provided by Regeneron Pharmaceuticals); only the cells which expressed this construct extended processes in response to NT-3 (the neurotrophin ligand for TrkC). The second construct encoded the Green-Fluorescent Protein; cells expressing this construct fluoresced green. In this image of live cells, taken three days after biolistic co-transfection and treatment with NT-3, four cells expressed GFP. Notably, three of the four cells grew extensive processes in response to NT-3 treatment, while those cells which were not green were non-responsive. Such experiments illustrate the high efficiency of co-transfection using Biolistic bombardment.

Transfection of Organotypic Brain Slices
Brain Slice Culture Preparation
Under sterile conditions, 400 m-thick coronal slices of postnatal day 14 (P14) ferret and P0-P5 rat cortex are prepared as described elsewhere (McAllister, Lo, and Katz, 1995). We use a short-term interface culture system described originally by Stoppini et al., 1991, in which the slices are placed on Millicell inserts so that the top of the slice is exposed to the incubator atmosphere (5% CO₂ and 36 C) while the bottom of the slice contacts the culture medium. Although we generally assay these slices after 36 hours, if fed every 3 days with fresh media, they will continue to survive for weeks to months in culture, forming layer-specific connections (L.C.K., unpublished data). As soon as possible after slicing, we bombard the slices, then culture them for 36 hours before visualizing the transfected gene product.
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