%0 Journal Article
%A Marx, M.
%A Günter, R.H.
%A Hucko, W.
%A Radnikow, G.
%A Feldmeyer, D.
%T Improved biocytin labeling and neuronal 3D reconstruction
%J Nature protocols
%V 7
%@ 1754-2189
%C Basingstoke
%I Nature Publishing Group
%M PreJuSER-19855
%P 394 - 407
%D 2012
%Z 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.
%X 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.
%K Animals
%K Brain: cytology
%K Brain: ultrastructure
%K Imaging, Three-Dimensional: methods
%K Lysine: analogs & derivatives
%K Mice
%K Microtomy: methods
%K Neurons: ultrastructure
%K Osmium Tetroxide
%K Rats
%K Staining and Labeling: methods
%K Osmium Tetroxide (NLM Chemicals)
%K Lysine (NLM Chemicals)
%K biocytin (NLM Chemicals)
%K J (WoSType)
%F PUB:(DE-HGF)16
%9 Journal Article
%$ pmid:22301777
%U <Go to ISI:>//WOS:000300402300015
%R 10.1038/nprot.2011.449
%U https://juser.fz-juelich.de/record/19855