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
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Abstract

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

     
 
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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
     
 
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Materials

Basic Protocol 1: Bullet Preparation

  Materials
  • 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

  Materials
  • 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

  Materials
  • 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

  Materials
  • 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

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

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

  Materials
  • Imaris software package (Version 7.0, Bitplane)
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Figures

Videos

Literature Cited

Literature Cited
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