Basic Neuroanatomical Methods

Charles R. Gerfen1

1 National 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

  • 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 1: 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 protocol 1) or perfusion‐fixed (see 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 protocol 9)
  • 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 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 protocol 9)

Basic Protocol 5: Vibratome Sectioning

  Materials
  • Fresh‐frozen (unfixed) slide‐mounted brain sections (see 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 6: Post‐Sectioning Procedures I: Defatting

  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 7: Post‐Sectioning Procedures II: Thionin Staining

  Materials
  • Emulsion: Kodak NTB‐3 or Amersham LM‐1 (thaw 30 min prior to use)
  • 0.1% (w/v) Dreft detergent in H 2O
  • Slide‐mounted radioactively labeled tissue sections
  • Dektol developer (Kodak)
  • Stop bath: H 2O or 1.5% acetic acid in H 2O
  • 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)

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

  Materials
  • Gelatin‐subbing solution (see recipe)
  • Glass slides
  • Slide racks
  • 40°C glassware‐drying oven
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Figures

Videos

Literature Cited

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