High-contrast en bloc staining of neuronal tissue for field emission scanning electron microscopy
- 12 January 2012
- journal article
- research article
- Published by Springer Science and Business Media LLC in Nature Protocols
- Vol. 7 (2), 193-206
- https://doi.org/10.1038/nprot.2011.439
Abstract
Conventional heavy metal post staining methods on thin sections lend contrast but often cause contamination. To avoid this problem, we tested several en bloc staining techniques to contrast tissue in serial sections mounted on solid substrates for examination by Field Emission Scanning Electron Microscope (FESEM). Because FESEM section imaging requires that specimens have higher contrast and greater electrical conductivity than transmission electron microscope (TEM) samples, our technique utilizes osmium impregnation (OTO) to make the samples conductive while heavily staining membranes for segmentation studies. Combining this step with other classic heavy metal en bloc stains including uranyl acetate, lead aspartate, copper sulfate and lead citrate produced clean, highly contrasted TEM and SEM samples of insect, fish, and mammalian nervous system. This protocol takes 7–15 days to prepare resin embedded tissue, cut sections and produce serial section images.Keywords
This publication has 46 references indexed in Scilit:
- Wiring specificity in the direction-selectivity circuit of the retinaNature, 2011
- Structure–function studies of blood and air capillaries in chicken lung using 3D electron microscopyRespiratory Physiology & Neurobiology, 2010
- The three-dimensional micro- and nanostructure of the aortic medial lamellar unit measured using 3D confocal and electron microscopy imagingMatrix Biology, 2008
- Array Tomography: A New Tool for Imaging the Molecular Architecture and Ultrastructure of Neural CircuitsNeuron, 2007
- Automating the Collection of Ultrathin Serial Sections for Large Volume TEM ReconstructionsMicroscopy and Microanalysis, 2006
- Serial Block-Face Scanning Electron Microscopy to Reconstruct Three-Dimensional Tissue NanostructurePLoS Biology, 2004
- Block-staining tissues with potassium ferrocyanide-reduced osmium tetroxide and lead aspartate for electron microscopic radioautography.Journal of Histochemistry & Cytochemistry, 1984
- The use of osmium-thiocarbohydrazide-osmium (OTO) and ferrocyanide-reduced osmium methods to enhance membrane contrast and preservation in cultured cells.Journal of Histochemistry & Cytochemistry, 1984
- Imidazole-buffered osmium tetroxide: an excellent stain for visualization of lipids in transmission electron microscopyJournal of Molecular Histology, 1982
- Enhanced visualization of peripheral nerve and sensory receptors in the scanning electron microscope using cryofracture and osmium-thiocarbohydrazide-osmium impregnationJournal of Neurocytology, 1981