Estimating Cell Number in the Central Nervous System by Stereological Methods: The Optical Disector and Fractionator

Jay S. Charleston1

1 Institute of Neurotoxicology and Neurological Disorders and Shin Nippon Biological Laboratories U.S.A., Redmond, Washington
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 12.6
DOI:  10.1002/0471140856.tx1206s06
Online Posting Date:  May, 2001
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Abstract

This unit describes techniques, based on recent advances in stereological methods, to obtain unbiased estimates of total cell or synapse number in discrete structures of the central nervous system. They combine unbiased counting frames, unbiased systematic random sampling, and unbiased estimates of the structure volume to produce the final estimate of number.

     
 
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Table of Contents

  • Basic Protocol 1: Estimating Total Particle Number Using Optical Fractionator
  • Alternate Protocol 1: Estimating Total Particle Number Using Nv,Vref
  • Alternate Protocol 2: Estimating Total Practical Number Using the Physical Dissector
  • Support Protocol 1: Glycolmethacrylate Embedding
  • Support Protocol 2: Giemsa Stain Procedure for Glycolmethacrylate Sections
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Estimating Total Particle Number Using Optical Fractionator

  Materials
  • Test animal
  • 4% (w/v) paraformaldehyde (see recipe), prepare fresh
  • Razor blades
  • Humidified chamber: Petri dish containing moistened filter paper or compartmented boxes (e.g., Brain Research Laboratories) filled with buffer
  • Sharp dissecting pins
  • Hand‐operated microtome (e.g., Sorvall JB‐4 or Reichert‐Jung 820‐II) with glass knives or disposable microtome razor blades (e.g., MB35, Shandon)
  • Frosted glass microscope slides (acid‐cleaned in 5 N HCl for 2 min, then rinsed in H 2O)
  • 70°C heated slide tray
  • Optical disector microscope
  • Tabletop linear tracking device (e.g., Microcator, Heidenhain)
  • Additional reagents and equipment for perfusion fixation (unit 9.5), glycolmethacrylate embedding (see protocol 4) or cryosectioning (see appendix 3A for reference) and Giemsa staining (see protocol 5)

Alternate Protocol 1: Estimating Total Particle Number Using Nv,Vref

  • One of the following setups for performing physical disector:
  •  Camera lucida for attachment to microscope, acetate sheets, and water‐soluble colored markers
  •  Two projecting microscopes in darkened room
  •  Commercial software/video system for capture and alignment of sequential section tracings (e.g., StereoInvestigator, MicroBrightField Bioquant, R&M Biometrics)

Alternate Protocol 2: Estimating Total Practical Number Using the Physical Dissector

  Materials
  • Fixed specimen
  • 50%, 70%, 90%, and 95% ethanol
  • 50% (v/v) ethanol/glycolmethacrylate solution
  • 100% glycolmethacrylate (e.g., Jung Historesin; Leica Instruments)
  • Polymerizer
  • Embedding molds (e.g., EBH block holder and molds; Polyscience)

Support Protocol 1: Glycolmethacrylate Embedding

  Materials
  • Giemsa blood staining stock solution (Baker Analyzed, e.g., Fisher)
  • 2% (v/v) acetic acid
  • 50%, 70%, 90%, 95%, and 100% ethanol
  • 100% xylene
  • High‐viscosity mounting medium (e.g., Cytoseal 280, Stephens Scientific)
  • 60°C water bath
  • Coverslips
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Figures

Videos

Literature Cited

Literature Cited
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