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

Tapia, JC; Kasthuri, N; Hayworth, KJ; Schalek, R; Lichtman, JW; Smith, SJ; Buchanan, J.

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.

Más información

Título según WOS: High-contrast en bloc staining of neuronal tissue for field emission scanning electron microscopy
Título de la Revista: NATURE PROTOCOLS
Volumen: 7
Número: 2
Editorial: Nature Publishing Group
Fecha de publicación: 2012
Página de inicio: 193
Página final: 206
Idioma: English
DOI:

10.1038/nprot.2011.439

Notas: ISI