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000019855 084__ $$2WoS$$aBiochemical Research Methods
000019855 1001_ $$0P:(DE-Juel1)VDB6549$$aMarx, M.$$b0$$uFZJ
000019855 245__ $$aImproved biocytin labeling and neuronal 3D reconstruction
000019855 260__ $$aBasingstoke$$bNature Publishing Group$$c2012
000019855 300__ $$a394 - 407
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000019855 440_0 $$024802$$aNature Protocols$$v7
000019855 500__ $$aThis work was supported by the Deutsche Forschungsgemeinschaft (DFG; Research Group BaCoFun), the Helmholtz Association and the Helmholtz Alliance for Systems Biology. We thank A. Rollenhagen for help with the EM protocol and T. Abel for critically reading the manuscript.
000019855 520__ $$aIn 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.
000019855 536__ $$0G:(DE-Juel1)FUEK409$$2G:(DE-HGF)$$aFunktion und Dysfunktion des Nervensystems (FUEK409)$$cFUEK409$$x0
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000019855 650_2 $$2MeSH$$aAnimals
000019855 650_2 $$2MeSH$$aBrain: cytology
000019855 650_2 $$2MeSH$$aBrain: ultrastructure
000019855 650_2 $$2MeSH$$aImaging, Three-Dimensional: methods
000019855 650_2 $$2MeSH$$aLysine: analogs & derivatives
000019855 650_2 $$2MeSH$$aMice
000019855 650_2 $$2MeSH$$aMicrotomy: methods
000019855 650_2 $$2MeSH$$aNeurons: ultrastructure
000019855 650_2 $$2MeSH$$aOsmium Tetroxide
000019855 650_2 $$2MeSH$$aRats
000019855 650_2 $$2MeSH$$aStaining and Labeling: methods
000019855 650_7 $$020816-12-0$$2NLM Chemicals$$aOsmium Tetroxide
000019855 650_7 $$056-87-1$$2NLM Chemicals$$aLysine
000019855 650_7 $$0576-19-2$$2NLM Chemicals$$abiocytin
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000019855 7001_ $$0P:(DE-Juel1)VDB98246$$aGünter, R.H.$$b1$$uFZJ
000019855 7001_ $$0P:(DE-Juel1)VDB104574$$aHucko, W.$$b2$$uFZJ
000019855 7001_ $$0P:(DE-Juel1)VDB36676$$aRadnikow, G.$$b3$$uFZJ
000019855 7001_ $$0P:(DE-Juel1)131680$$aFeldmeyer, D.$$b4$$uFZJ
000019855 773__ $$0PERI:(DE-600)2244966-8$$a10.1038/nprot.2011.449$$gVol. 7, p. 394 - 407$$p394 - 407$$q7<394 - 407$$tNature protocols$$v7$$x1754-2189$$y2012
000019855 8567_ $$uhttp://dx.doi.org/10.1038/nprot.2011.449
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