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DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; Gan et al., 2000). Here we provide a detailed description of each step required together with exemplary images of good and bad outcomes that will help when setting up the technique. In our experience, a few steps proved critical for the successful application of DiOLISTICS. These considerations include the quality of the DiI-coated bullets, the extent of fixative exposure, and the concentration of detergent used in the incubation solutions. Tips and solutions for common problems are provided.
This is a versatile labeling technique that can be applied to multiple animal species at a wide range of ages. Unlike other fluorescent labeling techniques that are limited to preparations from young animals or restricted to mice because they rely on the expression of a fluorescent transgene, DiOLISTIC labeling can be applied to animals of all ages, species and genotypes and it can be used in combination with immunostaining to identify a specific subpopulation of cells. Here we demonstrate the use of DiOLISTICS to label neurons in brain slices from adult mice and adult non-human primates with the purpose of quantifying dendrite branching and dendritic spine morphology.
1. Preparing DiI/Tungsten Bead Bullets: DiI (1-1'-Dioctadecyl-3,3,3',3'- tetramethylindocarbocyanine perchlorate) bullets should be prepared in advance of cutting brain slices. The procedure takes approximately 2-4 hours depending on how many bullets are prepared at once.
2. Preparing polyvinylpyrrolidone (PVP) solution and tubing: To improve bead attachment to the bullet tubing (TEZFEL tubing), coat it with polyvinylpyrrolidone (PVP) solution.
3. DiI Staining: This labeling technique can be used to stain neurons in brain tissue from diverse species; in this particular case, from mouse (3-6 month old) and non-human primates (9-15 years old).
It is preferable that slices are prepared from fixed brain tissue obtained by transcardiac perfusion of fixative solution because tissue preservation is expected to be best under this condition. However, fixation by perfusion is not always feasible (as was the case with the monkey tissue) and DiOLISTIC labeling can still be successfully applied under different procedures for tissue preparation. Here we describe three different procedures for tissue preparation:
In the present study, the monkey brains were obtained from subjects that were transcardially perfused with ice cold artificial cerebral spinal fluid (ACSF) prior to brain harvest and a block (4 mm thick) of tissue containing the caudate and putamen was fixed for 60 min at room temperature. For all procedures, fixative solution contains 4% paraformaldehyde and 4% sucrose in PBS (prepared fresh or used within 15 days). It is important to note that fixation times are critical for successful staining. Overfixation can affect staining by disrupting the integrity of the plasma membrane and causing dye to leak out of the cell (see more information under results).
4. Antibody Staining: Immunostaining should be performed after step 3.4 and before mounting.
5. Representative Results:
Figure 1. DiI labeling of neurons in brain slices from non-human primates. (A) Low magnification image of a stained brain slice containing the caudate nucleus from a cynomolgus monkey that shows sparse labeling of neurons with DiI. (B-C) Isolated medium spiny neurons can be easily identified using a fluorescent dissecting scope.
Figure 2. Troubleshooting DiOLISTIC labeling. (A-B) Stained slices from dorsal striatum of monkey (A) and mouse (B) showing large clumps of dye-coated beads and as a consequence, no individual cellular elements can be distinguished. (C-D) Images that exemplify the result of applying DiOLISTIC labeling to sections that were kept in fixative solution for prolonged periods of time or where tissue preservation failed in monkey (C) and mouse (D) tissue.
Figure 3. Confocal image of striatal medium spiny neuron stained with DiI. (A), Image stack of a whole cell allows for morphological analysis of the dendritic branches. (B-C) Dendrite segments from mouse (B) and monkey (C) medium spiny neurons show clear labeling of dendrite and spines.
Figure 4. Combining DiOLISTIC labeling with immunostaining. (A) DiI labeledmedium spiny neuron from striatum of BAC transgenic mouse expressing GFP under the D1 dopamine receptor promoter. (B) Immunostaining using anti-GFP antibody as described by methods adapted from Lee et al. (2006). Same field as A. (C) Composite image of red and green fluorescence identifies DiI-labeled neuron as a GFP-positive neuron.
DiOLISTIC labeling is one of the most versatile techniques available for fluorescently labeling cells because it can be applied to tissue sections from diverse species, to a wide range of ages, and to tissue obtained fresh or from fixative-perfused animals (see also Gan et al., 2009). The process is relatively fast as it takes 1-2 days and it can be combined with other more classical labeling approaches such as immunostaining (Lee et al., 2006). Special attention should be paid to avoid over fixing and the use of high detergent concentrations in incubation solutions because these will comprise the integrity of the lipophilic membrane and cause the dye to leak out of the cells. Antibody penetration can be facilitated by low concentrations of Triton X-100 (Lee et al., 2006), as well as digitonin or saponin in the incubation solution (Matsubayashi et al., 2008). Some modifications that can be applied to this technique include the use of bullets with multiple dyes as described by Gan et al. (2000) or changing the length and type of antibody exposure (Neely et al., 2009).
Traditionally, DiI has been used to trace neuronal projections in the brain. DiOLISTIC labeling expands its application by describing a method useful to examine cell morphology. Neuronal morphology is of great interest because of the large diversity found in the brain and the speculation that cell shape might reflect on the function multiplicity of neuronal populations in the nervous system. One example of this is the fact that many neurons in the mammalian nervous system display small protrusion called dendritic spines that are the site of glutamatergic synapses. In this way, the density of glutamatergic synapses onto a cell can be correlated to the density of dendritic spines, which can be measured using the labeling technique described herein. Furthermore, other morphological parameters such as dendrite total length, branching pattern, dendritic spine shape and density can be quantified and studied.
The use of fluorescent labeling for studying neuronal morphology has many advantages when compared to more traditional techniques that rely on bright field microscopy (e.g. Golgi staining) because it allows for higher resolution confocal imaging. Another advantage of using DiI as a fluorophore in experiments aimed at measuring dendritic spine density and morphology is its lipophilic properties. DiI partitions into the plasma membrane and provides a well defined outline of the neuronal processes and dendritic protrusions. Given the small volume of most dendritic spines (less than 1 femtoliter), membrane staining is more efficient and allows for better visualization of small, thin protrusions than cytoplasmic staining.
All animal procedures were performed following guidance from the Animal Care and Use Committee at NIAAA and the Oregon National Primate Research Center. The authors have nothing to disclose.
We would like to acknowledge Michael Feyder and Terrell Holloway for their assistance during the initial set up of the technique and Dr. Fumi Ono s laboratory for the access to their confocal microscope. This research was funded by the National Institute of Health through the intramural program of NIAAA and NINDs.