Improved biocytin labeling and neuronal 3D reconstruction

被引：64

作者：

Marx, Manuel ^{[1
]}

Guenter, Robert H. ^{[1
]}

Hucko, Werner ^{[1
]}

Radnikow, Gabriele ^{[1
]}

Feldmeyer, Dirk ^{[1
,2
,3
]}

机构：

[1] Res Ctr Julich, Inst Neurosci & Med INM 2, Julich, Germany

[2] Rhein Westfal TH Aachen, Dept Psychiat Psychotherapy & Psychosomat, Aachen, Germany

[3] Julich Aachen Res Alliance Translat Brain Med JAR, Aachen, Germany

来源：

NATURE PROTOCOLS | 2012年 / 7卷 / 02期

关键词：

RAT BARREL CORTEX; ANATOMICALLY IDENTIFIED BASKET; SOMATOSENSORY CORTEX; PYRAMIDAL NEURONS; IN-VITRO; SYNAPTIC CONNECTIONS; ELECTRON-MICROSCOPY; CYTOCHROME-OXIDASE; INHIBITORY NEURONS; CEREBRAL-CORTEX;

D O I：

10.1038/nprot.2011.449

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

In this report, we describe a reliable protocol for biocytin labeling of neuronal tissue and diaminobenzidine (DAB)-based processing of brain slices. We describe how to embed tissues in different media and how to subsequently histochemically label the tissues for light or electron microscopic examination. We provide a detailed dehydration and embedding protocol using Eukitt that avoids the common problem of tissue distortion and therefore prevents fading of cytoarchitectural features (in particular, lamination) of brain tissue; as a result, additional labeling methods (such as cytochrome oxidase staining) become unnecessary. In addition, we provide correction factors for tissue shrinkage in all spatial dimensions so that a realistic neuronal morphology can be obtained from slice preparations. Such corrections were hitherto difficult to calculate because embedding in viscous media resulted in highly nonlinear tissue deformation. Fixation, immunocytochemistry and embedding procedures for light microscopy (LM) can be completed within 42-48 h. Subsequent reconstructions and morphological analyses take an additional 24 h or more.

引用

页码：394 / 407

页数：14

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