TY  - JOUR
AU  - Marx, M.
AU  - Günter, R.H.
AU  - Hucko, W.
AU  - Radnikow, G.
AU  - Feldmeyer, D.
TI  - Improved biocytin labeling and neuronal 3D reconstruction
JO  - Nature protocols
VL  - 7
SN  - 1754-2189
CY  - Basingstoke
PB  - Nature Publishing Group
M1  - PreJuSER-19855
SP  - 394 - 407
PY  - 2012
N1  - This work was supported by the Deutsche Forschungsgemeinschaft (DFG; Research Group BaCoFun), the Helmholtz Association and the Helmholtz Alliance for Systems Biology. We thank A. Rollenhagen for help with the EM protocol and T. Abel for critically reading the manuscript.
AB  - 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.
KW  - Animals
KW  - Brain: cytology
KW  - Brain: ultrastructure
KW  - Imaging, Three-Dimensional: methods
KW  - Lysine: analogs & derivatives
KW  - Mice
KW  - Microtomy: methods
KW  - Neurons: ultrastructure
KW  - Osmium Tetroxide
KW  - Rats
KW  - Staining and Labeling: methods
KW  - Osmium Tetroxide (NLM Chemicals)
KW  - Lysine (NLM Chemicals)
KW  - biocytin (NLM Chemicals)
KW  - J (WoSType)
LB  - PUB:(DE-HGF)16
C6  - pmid:22301777
UR  - <Go to ISI:>//WOS:000300402300015
DO  - DOI:10.1038/nprot.2011.449
UR  - https://juser.fz-juelich.de/record/19855
ER  -