Dendritic spines observed by extracellular DiI dye and immunolabeling under confocal microscopy

Different methods have been used for over a century to study and quantify dendritic spines, which are specialized postsynaptic compartments whose shape and density change on natural or pathological conditions. Here, we describe the steps to apply the sonicated fine powdered carbocyanine dye DiI extracellularly and with immunolabeling of synaptic proteins on brain slices of adult rats. This approach reveals detailed features of dendritic spine morphology, number, distribution, and connectivity under confocal microscopy. The classic Golgi-impregnation was done to serve as comparison. Sonicated powdered DiI labeling is easier than other methodologies with advantages over full DiI crystal applications or other intracellular dyes. This is a quick and reproducible procedure to fluorescently label spines for 3-dimensional imaging. The method can be used together with the co-immunolocalization of synaptic proteins.