Article

High contrast en bloc staining of neuronal tissue for field emission scanning electron microscopy

Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, USA.
Nature Protocol (Impact Factor: 8.36). 02/2012; 7(2):193-206. DOI: 10.1038/nprot.2011.439
Source: PubMed

ABSTRACT Conventional heavy metal poststaining 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 microscopy (FESEM). Because FESEM section imaging requires that specimens have higher contrast and greater electrical conductivity than transmission electron microscopy (TEM) samples, our technique uses 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 (UA), lead aspartate, copper sulfate and lead citrate, produced clean, highly contrasted TEM and scanning electron microscopy (SEM) samples of insect, fish and mammalian nervous systems. This protocol takes 7-15 d to prepare resin-embedded tissue, cut sections and produce serial section images.

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Available from: Narayanan Kasthuri, Jan 27, 2015
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    • "For large volumes to be imaged quickly, good contrast is essential. We often use a combination of the (R)OTO technique for enhancing osmium staining en bloc and lead citrate post section staining (Tapia et al., 2012). It is important to note, that by thickening and darkening membranes, this technique can make synapses more difficult to identify in single sections. "
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    • "Recently, methods for efficient ultrastructural imaging of large volumes have been developed that are based on the automated sectioning of a tissue block (Denk and Horstmann, 2004; Hayworth et al., 2006; Kasthuri et al., 2007; Helmstaedter et al., 2008; Knott et al., 2008; Briggman and Bock, 2012; Denk et al., 2012). In one approach, an automated tape-collecting ultramicrotome (ATUM) is used to cut sections at a thickness of <30 nm and collect them on a carbon-coated tape (Hayworth et al., 2006; Kasthuri et al., 2007; Tapia et al., 2012). Sections are then imaged in a scanning electron microscope (SEM). "
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    • "Since the biological specimen consisted of carbon, oxygen, hydrogen etc which are less electron dense elements, thus produced micrographs are of moderate contrast. In order to overcome this problem, heavy metal salt (uranyl acetate, lead citrate, phosphotangstic acid, ammonium molybdate etc.) staining methods are employed to elucidate better the ultrastructural details of the sample under observation [23] [24] [25] [26] [27] [28]. The arrangement of cells, their polarization status, the connective tissues networks within various tissue samples etc.in 2D can be vividly examined by TEM. "
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