DiOlistic Labeling in Fixed Brain Slices: Phenotype, Morphology, and Dendritic Spines

Nancy A. Staffend1, Robert L. Meisel1

1 Department of Neuroscience, University of Minnesota, Minneapolis, Minnesota
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 2.13
DOI:  10.1002/0471142301.ns0213s55
Online Posting Date:  April, 2011
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


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. Curr. Protoc. Neurosci. 55:2.13.1‐2.13.15. © 2011 by John Wiley & Sons, Inc.

Keywords: DiOlistic; gene gun; dendritic spine; neuronal morphology

PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Bullet Preparation
  • Basic Protocol 2: Tissue Preparation
  • Basic Protocol 3: Delivery of DiI‐Coated Tungsten Particles and Tissue Mounting
  • Alternate Protocol 1: Immunofluorescence
  • Basic Protocol 4: Confocal Imaging
  • Alternate Protocol 2: Quantitation and Analysis of Dendritic Spine Density and Spine Head Morphology
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
PDF or HTML at Wiley Online Library


Basic Protocol 1: Bullet Preparation

  • 10 mg/ml polyvinylpyrrolidone (PVP; Sigma‐Aldrich) dissolved in deionized water
  • Nitrogen gas/regulator
  • Carbocyanine fluorescent DiI or CM‐DiI (Molecular Probes)
  • Methylene chloride
  • 1.3‐µm tungsten particles (BioRad)
  • Desiccant
  • Tefzel tubing (BioRad)
  • Microcentrifuge tubes
  • Glass slides
  • Single‐edged razor blades
  • Bath sonicator
  • Vortex
  • 25‐ml syringes

Basic Protocol 2: Tissue Preparation

  • Animal
  • Anesthetic—specific to animal (requires animal protocol from specific institute)
  • Phosphate‐buffered saline (PBS; see recipe), pH 7.4
  • 1.5% (w/v) paraformaldehyde in PBS (see recipe)
  • Cyanoacrylate glue
  • Surgical scissors
  • Perfusion pump equipped with Tygon laboratory tubing (R‐3603; Saint‐Gobain Performance Plastics)
  • 1‐ml syringes equipped with 26‐G needles
  • Rongeurs
  • Brain matrix (available from several suppliers sized for either rat or mouse brains)
  • Single‐edged razor blades
  • Mounting block
  • 35‐mm tissue culture dishes
  • Vibratome
  • Vibratome blades
  • Paint brush
NOTE: All protocols using live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) and must follow officially approved procedures for the care and use of laboratory animals.

Basic Protocol 3: Delivery of DiI‐Coated Tungsten Particles and Tissue Mounting

  • DiI bullets (see protocol 1)
  • Helium gas/regulator
  • 150‐ or 300‐µm thick tissue slices (see protocol 2)
  • 25 mM PBS, pH 7.2 (see recipe)
  • 4% (w/v) paraformaldehyde in PBS (see recipe)
  • 5% (w/v) n‐propyl‐gallate in glycerin (or any other anti‐fade agent may be used)
  • Helios gene gun (BioRad) with accessories
  • Modified barrel (O'Brien et al., )
  • 40‐mm spacer (O'Brien et al., )
  • 70‐µm nylon mesh filter (Plastok Associates)
  • Helium hose assembly with quick release fitting to connect to the gene gun
  • 5‐ml disposable plastic pipets
  • Superfrost slides (Brain Research Laboratories)
  • Coverslips

Alternate Protocol 1: Immunofluorescence

  • DiI‐labeled tissue section (see protocol 3)
  • 0.1% and 0.01% Triton‐X 100 (see recipe)
  • 0.1% and 10% bovine serum albumin (BSA; see reciperecipes)
  • Nail polish
  • Primary antibody of interest
  • Appropriate fluorescent secondary antibody for conjugation—ensure secondary antibody and DiI excitation/emission spectra do not overlap
  • 5% (w/v) n‐propyl‐gallate in glycerin
  • Superfrost slides (Brain Research Laboratories)
  • Coverslips

Basic Protocol 4: Confocal Imaging

  • Confocal microscope with 20×, 63×, and 100× objectives

Alternate Protocol 2: Quantitation and Analysis of Dendritic Spine Density and Spine Head Morphology

  • Imaris software package (Version 7.0, Bitplane)
PDF or HTML at Wiley Online Library



Literature Cited

Literature Cited
   Buhl, E.H. and Lubke, J. 1989. Intracellular lucifer yellow injection in fixed brain slices combined with retrograde tracing, light and electron microscopy. Neuroscience 28:3‐16.
   Chklovskii, D.B. 2004. Synaptic connectivity and neuronal morphology: Two sides of the same coin. Neuron 43:609‐617.
   Coss, R.G. and Perkel, D.H. 1985. The function of dendritic spines: a review of theoretical issues. Behav. Neural Biol. 44:151‐185.
   Cui, Z.J., Zhao, K.B., Zhao, H.J., Yu, D.M., Niu, Y.L., Zhang, J.S., and Deng, J.B. 2010. Prenatal alcohol exposure induces long‐term changes in dendritic spines and synapses in the mouse visual cortex. Alcohol Alcohol. 45:312‐319.
   Fairen, A. 2005. Pioneering a golden age of cerebral microcircuits: The births of the combined Golgi‐electron microscope methods. Neuroscience 136:607‐614.
   Forlano, P.M. and Woolley, C.S. 2010. Quantitative analysis of pre‐ and postsynaptic sex differences in the nucleus accumbens. J. Comp. Neurol. 518:1330‐1348.
   Gan, W.B. and Lichtman, J.W. 1998. Synaptic segregation at the developing neuromuscular junction. Science 282:1508‐1511.
   Gan, W.B., Bishop, D.L., Turney, S.G., and Lichtman, J.W. 1999. Vital imaging and ultrastructural analysis of individual axon tertminals labeled by iontophoretic application of lipophilic dye. J. Neurosci. Methods 93:13‐20.
   Gan, W.B., Grutzendler, J., Wong, W.T., Wong, R.O., and Lichtman, J.W. 2000. Multicolor “DiOlistic” labeling of the nervous system using lipophilic dye combinations. Neuron 27:219‐225.
   Hollingworth, T. and Berry, M. 1975. Network analysis of dendritic fields of pyramidal cells in neocortex and Purkinje cells in the cerebellum of the rat. Philos. Trans. R. Soc. Lond. B Biol. Sci. 270:227‐264.
   Honig, M.G. and Hume, R.I. 1986. Fluorescent carbocyanine dyes allow living neurons of identified origin to be studied in long‐term cultures. J. Cell Biol. 103:171‐187.
   Kasai, H., Fukuda, M., Watanabe, S., Hayashi‐Takagi, A., and Noguchi, J. 2010. Structural dynamics of dendritic spines in memory and cognition. Trends Neurosci. 33:121‐129.
   Li, M., Cui, Z., Niu, Y., Liu, B., Fan, W., Yu, D., and Deng, J. 2010. Synaptogenesis in the developing mouse visual cortex. Brain Res. Bull. 81:107‐113.
   Liu, D.W. and Westerfield, M. 1990. The formation of terminal fields in the absence of competitive interactions among primary motoneurons in the zebrafish. J. Neurosci. 10:3947‐3959.
   Moolman, D.L., Vitolo, O.V., Vonsattel, J.P., and Shelanski, M.L. 2004. Dendrite and dendritic spine alterations in Alzheimer models. J. Neurocytol. 33:377‐387.
   Neely, M.D., Stanwood, G.D., and Deutch, A.Y. 2009. Combination of diOlistic labeling with retrograde tract tracing and immunohistochemistry. J. Neurosci. Methods 184:332‐336.
   O'Brien, J.A., Holt, M., Whiteside, G., Lummis, S.C., and Hastings, M.H. 2001. Modifications to the hand‐held Gene Gun: Improvements for in vitro biolistic transfection of organotypic neuronal tissue. J. Neurosci. Methods 112:57‐64.
   O'Rourke, N.A., Cline, H.T., and Fraser, S.E. 1994. Rapid remodeling of retinal arbors in the tectum with and without blockade of synaptic transmission. Neuron 12:921‐934.
   Oberheim, N.A., Tian, G.F., Han, X., Peng, W., Takano, T., Ransom, B., and Nedergaard, M. 2008. Loss of astrocytic domain organization in the epileptic brain. J. Neurosci. 28:3264‐3276.
   Purves, D. and Hume, R.I. 1981. The relation of postsynaptic geometry to the number of presynaptic axons that innervate autonomic ganglion cells. J. Neurosci. 1:441‐452.
   Russo, S.J., Dietz, D.M., Dumitriu, D., Morrison, J.H., Malenka, R.C., and Nestler, E.J. 2010. The addicted synapse: Mechanisms of synaptic and structural plasticity in nucleus accumbens. Trends Neurosci. 33:267‐276.
   Spacek, J. 1989. Dynamics of the Golgi method: A time‐lapse study of the early stages of impregnation in single sections. J. Neurocytol. 18:27‐38.
   Spergel, D.J., Kruth, U., Shimshek, D.R., Sprengel, R., and Seeburg, P.H. 2001. Using reporter genes to label selected neuronal populations in transgenic mice for gene promoter, anatomical, and physiological studies. Prog. Neurobiol. 63:673‐686.
   Wu, C.C., Reilly, J.F., Young, W.G., Morrison, J.H., and Bloom, F.E. 2004. High‐throughput morphometric analysis of individual neurons. Cereb. Cortex 14:543‐554.
   Wu, G.Y. and Cline, H.T. 1998. Stabilization of dendritic arbor structure in vivo by CaMKII. Science 279:222‐226.
PDF or HTML at Wiley Online Library