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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We thank the following foundations, companies and support funding for this work: the Gatsby Charitable Trust; a Howard Hughes Medical Institute (HHMI) Collaborative Research Award; the John S. McDonnell Foundation; the McKnight Foundation; the Mathers Foundation; the Center for Brain Science, Harvard; the Initiative for Innovative Computing; Microsoft Research; Carl Zeiss SMT; JEOL; and Fibics. We appreciate helpful discussions with W. Denk, J. Heuser, T. Reese and M.J. Karnovsky. We acknowledge N. Ghori and E. Hartwig for technical support, K. Micheva and G. O'Brien for comments on the manuscript, C. Genoud and J. Mancuso from Gatan for SBF-SEM imaging of zebrafish samples and R. Giberson of Ted Pella Inc. for advice on microwave conditions.
The authors declare no competing financial interests.
TEM images of (a) adult Drosophila brain and (b) L3 larval neuromuscular junction. Osmid/OTO /copper sulfate lead citrate en bloc staining 1 hour at 37° C. Scale bar is 2 µm in a and 1 µm in b. Animal use in this experiment was conducted in strict accordance to our institutional animal care and use committee guidelines. (TIFF 6454 kb)
TEM images of (a) 10 dpf larval Zebrafish optic tectum synapses en bloc stained Osmid/OTO copper sulfate lead citrate overnight at 37°C. Scale bar 0.2 µm (b) Synaptic vesicles are apparent in neuronal processes. Copper sulfate lead citrate en bloc overnight @ 25° C. Scale bar 0.5 µm. Animal use in this experiment was conducted in strict accordance to our institutional animal care and use committee guidelines. (TIFF 6533 kb)
Comparison of different en bloc staining conditions in TEM images of mouse cortex. (a) Longer incubations times with TCH cause intense darkening of cytoplasm. (b) Osmium imidazole and 2% aqueous uranyl acetate shows high contrast membranes but murky cytoplasm. (c) Osmium imidazole without TCH and en bloc UA shows poor contrast. (d) Osmium imidazole without TCH and with copper sulfate lead citrate en bloc shows pale cytoplasm and weak membrane staining. (e) Osmium imidazole, TCH and ethanolic phosphotungstic acid but no copper sulfate/lead citrate shows pale cytoplasm, intermediate membrane contrast and strongly stained post-synaptic densities. (f) Osmium imidazole without copper sulfate lead citrate staining and inadequate rinsing shows dense and murky cytoplasm. Scale bars 1 µm in b, d, e, f and 2 µm in a, c. Animal use in this experiment was conducted in strict accordance to our institutional animal care and use committee guidelines. (TIFF 8311 kb)
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Tapia, J., Kasthuri, N., Hayworth, K. et al. High-contrast en bloc staining of neuronal tissue for field emission scanning electron microscopy. Nat Protoc 7, 193–206 (2012). https://doi.org/10.1038/nprot.2011.439
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