High-resolution fluorescent labeling of living cerebellar slices

In the present study, we extend previous research on staining of living brain slices with fluorescent phospholipids. This new procedure allows high-resolution staining of specific cell types, in particular, Purkinje cells, in the cerebellar slice while not affecting the intrinsic electrical activity of the tissue. Four different nitrobenzoxadiole (NBD)-phospholipids were incorporated into living cerebellar slices via loading from small unilamellar vesicles (SUVs), composed of a carrier and the fluorescent lipid. The labeled acidic phospholipid, NBD-phosphatidic acid (NBD-PA), produced the highest resolution images with exquisite labeling of the dendritic fields. The label was incorporated predominantly into the Purkinje cell body (excluding the nucleus), with more diffuse staining in other cell types, including stellate, basket and granule cells. The labeled lipid concentration and composition of the carrier lipid were significant in determining the specificity of labeling. Labeling, which was optimal after a 1 h incubation, was present throughout the depth of the slice. This procedure provides a promising approach to fluorescent labeling that will allow simultaneous monitoring of changes in cellular morphology and electrophysiology of living brain slices.