title: High-contrast en bloc staining of neuronal tissue for field emission scanning electron microscopy.
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.
authors.affiliation: Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, USA.
authors.forename: Juan Carlos
authors.initials: JC
authors.lastname: Tapia
authors.forename: Narayanan
authors.initials: N
authors.lastname: Kasthuri
authors.forename: Kenneth J
authors.initials: KJ
authors.lastname: Hayworth
authors.forename: Richard
authors.initials: R
authors.lastname: Schalek
authors.forename: Jeff W
authors.initials: JW
authors.lastname: Lichtman
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Smith
authors.forename: JoAnn
authors.initials: J
authors.lastname: Buchanan
mesh_terms: D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry; D001921:Brain / Q000648:ultrastructure; D002462:Cell Membrane / Q000648:ultrastructure; D019343:Citric Acid / Q000737:chemistry; D019327:Copper Sulfate / Q000737:chemistry; D004330:Drosophila / Q000648:ultrastructure; D007854:Lead / Q000737:chemistry; D051379:Mice; D008855:Microscopy, Electron, Scanning / Q000379:methods*; D008867:Microtomy; D009417:Nerve Tissue / Q000648:ultrastructure*; D009942:Organometallic Compounds / Q000737:chemistry; D009992:Osmium / Q000737:chemistry; D013194:Staining and Labeling / Q000379:methods*; D015027:Zebrafish
chemical_list: D009942:Organometallic Compounds; C005460:uranyl acetate; D019343:Citric Acid; D009992:Osmium; D007854:Lead; D001224:Aspartic Acid; D019327:Copper Sulfate