This title appears in the Scientific Report :
2012
Please use the identifier:
http://dx.doi.org/10.1038/nprot.2011.449 in citations.
Improved biocytin labeling and neuronal 3D reconstruction
Improved biocytin labeling and neuronal 3D reconstruction
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 examin...
Saved in:
Personal Name(s): | Marx, M. |
---|---|
Günter, R.H. / Hucko, W. / Radnikow, G. / Feldmeyer, D. | |
Contributing Institute: |
Molekulare Organisation des Gehirns; INM-2 |
Published in: | Nature protocols, 7 (2012) S. 394 - 407 |
Imprint: |
Basingstoke
Nature Publishing Group
2012
|
Physical Description: |
394 - 407 |
DOI: |
10.1038/nprot.2011.449 |
PubMed ID: |
22301777 |
Document Type: |
Journal Article |
Research Program: |
Connectivity and Activity Funktion und Dysfunktion des Nervensystems |
Series Title: |
Nature Protocols
7 |
Subject (ZB): | |
Publikationsportal JuSER |
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. |