Basic Neuroanatomical Methods
This unit covers some of the basic procedures that are common to a wide range of neuroanatomical protocols. Among the neuroanatomical protocols covered in subsequentunits are those for:
labeling specific neuronal cell types on the basis of their neurochemicalphenotype using immunohistochemical localization of antigenic sites on proteinsor peptides (
) or in situ hybridization histochemical localization of themessenger RNAs that encode such proteins or peptides (
localizing neurotransmitter receptor-binding sites (
combinations of the above methods that allow the connections betweenphenotypically characterized neurons to be visualized.While each of these procedures requires certain specific histologic treatments, they allshare some common methodologies. This unit provides some of the basic commonprotocols, as well as some standard staining protocols used as an adjunct to more specificneuroanatomical protocols.Most neuroanatomical procedures involve the following steps: (1) brain preparation, (2)sectioning the brain, (3) labeling and staining the brain sections, (4) application of thesections to microscope slides, and (5) microscopic analysis. The sequence of the steps forlabeling and staining the brain sections and applying sections to microscope slides willvary depending on the neuroanatomical method used. Procedures are provided for thepreparation of unfixed, fresh brain tissue (see Basic Protocol 1) as well as for perfusionfixation of animals resulting in fixed neural tissue (see Basic Protocol 2). A variety of methods for sectioning brains are described, including frozen sectioning in a cryostat (seeBasic Protocol 3), frozen sectioning with a microtome (see Basic Protocol 4), andsectioning with a vibratome (see Basic Protocol 5). The choice of sectioning methoddepends on how the brain has been prepared and what histochemical method is to be used.Methods for paraffin sectioning (see
CPMB UNIT 14.1
in this manual) orultramicrotome sectioning (Bolam, 1992) are not covered in this unit. Three post-section-ing procedures are provided: defatting of slide-mounted sections (see Basic Protocol 6),thionin staining of the sections (see Basic Protocol 7), and coating of slides withphotographic emulsion for autoradiography (see Basic Protocol 8). Finally, a procedureis described for subbing slides with gelatin, which is necessary in some protocols in orderfor the sections to adhere to the slides (see Support Protocol).
All protocols using live animals must first be reviewed and approved by anInstitutional Animal Care and Use Committee (IACUC) and must follow officiallyapproved procedures for care and use of laboratory animals.
BASIC PROTOCOL 1
PREPARATION OF UNFIXED FRESH-FROZEN BRAIN TISSUE
How the brain should be prepared for histologic processing depends on the specificneuroanatomical protocol to be used. Some protocols require the brain to be removedfrom the skull fresh—i.e., immediately following the killing of the animal—whereas otherprotocols require the brain to be perfused in situ with a fixative. The method of brainpreparation used depends in part on the neurochemical that is to be localized; how a brainis prepared determines, in turn, how the brain should subsequently be sectioned.Certain protocols require that the brain be removed from the skull fresh, without fixation.For example, autoradiographic localization of neurotransmitter receptor-binding sites is
Contributed by Charles R. Gerfen
Current Protocols in Neuroscience
(1997) 1.1.1-1.1.11Copyright © 1997 by John Wiley & Sons, Inc.