Coating particles with carbocyanine dyes

Imaging and reconstruction of developing neurons require cells that are labeled in a way that distinguishes them from their neighbors. This can be achieved with ballistic labeling, which refers to the delivery of a cell label by means of carrier particles (tungsten or gold) propelled from a pressurized gun. Ballistic delivery can reach many dispersed cells in one shot and can deploy a wide variety of cell markers to neurons in diverse preparations. The three most commonly used types of ballistic labels are carbocyanine dyes, dextran-conjugated fluorescent markers, and DNA plasmids. The primary advantage of ballistic labeling is that multiple dispersed cells can be labeled quickly in live or fixed tissue. This article describes a protocol for coating tungsten particles (∼1 µm in diameter) with carbocyanine dyes, which are widely used to label neurons in tissue and neural cells in suspension. These dyes are lipophilic and highly fluorescent within lipid bilayers. Because tissue damage worsens with the increasing pressure required for deeper bullet penetration, ballistic labeling of neurons is most effective when the target cells are near the surface of the preparation. This protocol was developed for labeling ganglion cells in retinal flat mounts.