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Basic Neuroanatomical Methods

Charles R. Gerfen1

1National Institutes of Mental Health, Bethesda, Maryland

Publication Name: 
Current Protocols in Neuroscience
Unit Number: 
Unit 1.1
DOI: 
10.1002/0471142301.ns0101s23
Online Posting Date: 
August, 2003
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Abstract

This unit covers some of the basic procedures that are common to a wide range of neuroanatomical protocols. Procedures are provided for the preparation of unfixed, fresh brain tissue as well as for perfusion fixation of animals resulting in fixed neural tissue. A variety of methods for sectioning brains are described, including frozen sectioning in a cryostat, frozen sectioning with a microtome, and sectioning with a vibratome. The choice of sectioning method depends on how the brain has been prepared and what histochemical method is to be used. Three post-sectioning procedures are provided: defatting of slide-mounted sections, thionin staining of the sections, and coating of slides with photographic emulsion for autoradiography. Finally, a procedure is described for subbing slides with gelatin, which is necessary in some protocols in order for the sections to adhere to the slides.

     
 
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Table of Contents

  • Unit Introduction
  • Basic Protocol 1: Preparation of Unfixed Fresh-Frozen Brain Tissue
  • Basic Protocol 2: Perfusion Fixation
  • Basic Protocol 3: Cryostat Sectioning of Frozen Brain Tissue
  • Basic Protocol 4: Sliding-Microtome Sectioning of Fixed Brain Tissue
  • Basic Protocol 5: Vibratome Sectioning
  • Basic Protocol 6: Post-Sectioning Procedures I: Defatting
  • Basic Protocol 7: Post-Sectioning Procedures II: Thionin Staining
  • Basic Protocol 8: Post-Sectioning Procedures III: Photographic-Emulsion Coating of Slide-Mounted Sections for Autoradiography
  • Support Protocol: Preparation of Gelatin-Subbed Microscope Slides
  • Reagents and Solutions
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Preparation of Unfixed Fresh-Frozen Brain Tissue

 Materials
  • Isopentane
  • Dry ice
  • Rat or mouse for study
  • Anesthetic
  • Dissection instruments:
  •     Scissors
  •     Spatula
  •     Forceps
  • Plexiglas or metal sieve-like basket and metal container large enough to hold it

Basic Protocol 2: Perfusion Fixation

 Materials
  • Saline (0.9% w/v NaCl), 4°C
  • Fixative solution for perfusion (see recipe), room temperature
  • Rat or mouse for study
  • Anesthetic
  • Sucrose-infiltration solution, 4°C (see recipe)
  • Peristaltic perfusion pump (e.g., Masterflex with variable-speed standard drives from Cole-Parmer)
  • Masterflex Tygon tubing (0.25-in.)
  • Blunt 13-G and 15-G hypodermic needles
  • Surgical instruments (Roboz Surgical or Fine Science Tools) including:
  •     Scalpel
  •     Scissors
  •     Clamps
  •     Hegenbarth clip-applying forceps
  •     Hemostats
  •     Bone rongeur

Basic Protocol 3: Cryostat Sectioning of Frozen Brain Tissue

 Materials
  • Dry ice (powdered or pellets)
  • Embedding matrix (M-1 from Shandon/Lipshaw or OCT compound from Miles Labs)
  • Brain tissue: fresh-frozen (see Basic Protocol 1) or perfusion-fixed (see Basic Protocol 2) and frozen on dry ice just prior to sectioning
  • Cryostat microtome
  • Specimen holder (cryostat chuck; metal platform for supporting specimen during sectioning)
  • Gelatin-subbed microscope slides (see Support Protocol)
  • Clean, soft paint brush (optional)
  • 40°C warming plate (optional)
  • Small zip-lock bags

Basic Protocol 4: Sliding-Microtome Sectioning of Fixed Brain Tissue

 Materials
  • Sucrose-infiltrated fixed brains (see Basic Protocol 2)
  • Dry ice
  • Potassium phosphate–buffered saline (KPBS; see recipe)
  • Sliding microtome with knife (Leica or American Optical) and sliding microtome stage
  • Small brush
  • Container for collecting brain tissue sections (e.g., 24-well Costar tissue culture plate)
  • Gelatin-subbed microscope slides (see Support Protocol)

Basic Protocol 6: Post-Sectioning Procedures I: Defatting

 Materials
  • Fresh-frozen (unfixed) slide-mounted brain sections (see Basic Protocol 3)
  • 4% formaldehyde in saline (see recipe)
  • Acetic anhydride
  • Triethanolamine/saline solution (see recipe)
  • 70%, 95%, and 100% ethanol
  • Chloroform
  • Metal 30-slide rack (optional for small numbers of slides)
  • 500-ml (or appropriate-sized) staining dishes

Basic Protocol 7: Post-Sectioning Procedures II: Thionin Staining

 Materials
  • Brain sections mounted on slides, preferably fixed
  • Thionin solution (see recipe)
  • 50%, 70%, 95%, and 100% ethanol
  • Xylene
  • 95% ethanol/1% acetic acid (optional)
  • Coverslips
  • Permount histological mounting fluid (e.g., Fisher)

Basic Protocol 8: Post-Sectioning Procedures III: Photographic-Emulsion Coating of Slide-Mounted Sections for Autoradiography

 Materials
  • Emulsion: Kodak NTB-3 or Amersham LM-1 (thaw 30 min prior to use)
  • 0.1% (w/v) Dreft detergent in H2O
  • Slide-mounted radioactively labeled tissue sections
  • Dektol developer (Kodak)
  • Stop bath: H2O or 1.5% acetic acid in H2O
  • Rapid Fix (Kodak): prepare according to manufacturer's instructions without hardener
  • Darkroom with amber/red sodium photographic safelight and humidifier
  • Slide mailer (2-slide or 5-slide, Shandon/Lipshaw or Thomas Scientific)
  • 40° to 42°C water bath
  • Blank microscope slides
  • Light-tight slide boxes
  • Staining racks and dishes
  • Glass staining dishes (Thomas Scientific)
  • Metal slide racks (Thomas Scientific)

Support Protocol: Preparation of Gelatin-Subbed Microscope Slides

 Materials
  • Gelatin-subbing solution (see recipe)
  • Glass slides
  • Slide racks
  • 40°C glassware-drying oven
Looking for materials?  Suppliers Guide
     
 
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Literature Cited

Literature Cited
    Bolam, J.P. (ed.) 1992. Experimental Neuroanatomy: A Practical Approach. Oxford University Press, Oxford.
     
 
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