Diolistic labeling in fixed brain slices: Phenotype, morphology, and dendritic spines

Identifying neuronal morphology is a key component in understanding neuronal function. Several techniques have been developed to address this issue, including Golgi staining, electroporation of fluorescent dyes, and transfection of fluorescent constructs. Ballistic delivery of transgenic constructs has been a successful means of rapidly transfecting a nonbiased population of cells within tissue or culture. Recently, this technique was modified for the ballistic delivery of dye-coated gold or tungsten particles, enabling a nonbiased, rapid fluorescent membrane labeling of individual neurons in both fixed and nonfixed tissue. This unit outlines a step-by-step protocol for the ballistic method of dye delivery ("DiOlistic" labeling) to fixed tissue, including optimal tissue fixation conditions. In addition, a protocol for coupling "DiOlistic" labeling with other immunofluorescent methods is detailed, enabling the association of neuronal morphology with a specific cellular phenotype.