Passa al contenuto
Merck

Improved biocytin labeling and neuronal 3D reconstruction.

Nature protocols (2012-02-04)
Manuel Marx, Robert H Günter, Werner Hucko, Gabriele Radnikow, Dirk Feldmeyer
ABSTRACT

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.

MATERIALI
N° Catalogo
Marchio
Descrizione del prodotto

Sigma-Aldrich
HEPES, ≥99.5% (titration)
Sigma-Aldrich
Citocromo c, ≥95% (SDS-PAGE)
Sigma-Aldrich
Osmium tetroxide, ReagentPlus®, 99.8%
Sigma-Aldrich
Osmium tetroxide solution, 4 wt. % in H2O
Sigma-Aldrich
Biocytin, ≥98% (TLC)
Sigma-Aldrich
Osmium tetroxide solution, suitable for electron microscopy, 4% in H2O
Sigma-Aldrich
Osmium tetroxide solution, 2.5 wt. % in tert-butanol
Sigma-Aldrich
Saccarosio, puriss., meets analytical specification of Ph. Eur., BP, NF
Sigma-Aldrich
Osmium tetroxide, ACS reagent, ≥98.0%
Sigma-Aldrich
Acido L-ascorbico, BioUltra, ≥99.5% (RT)
Sigma-Aldrich
Osmium tetroxide solution, suitable for electron microscopy, 2% in H2O
Sigma-Aldrich
Osmium tetroxide, Sealed ampule.
Sigma-Aldrich
Os EnCat® 40, extent of labeling: 0.3 mmol/g Os loading
Sigma-Aldrich
Anticorpo anti-fosfotirosina, clone 4G10®, coniugato con FITC, clone 4G10®, Upstate®, from mouse