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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.

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Figure 1: Preparation of serial thin sections.
Figure 2: LM photomicrograph of unstained 0.5-μm semithin section of epoxy-embedded mouse cortex shows crisp cellular details and good contrast.
Figure 3: Set of three representative FESEM images of 70-nm sections from a series of 25 on carbon-coated glass coverslips.
Figure 4: FESEM images of ROTO/en bloc lead aspartate staining of adult mouse brain tissue.
Figure 5: FESEM images of mouse cortex epoxy resin thin sections en bloc stained with Osmid, OTO and copper sulfate/lead citrate overnight at 20 °C.
Figure 6: Three-view block face FESEM of zebrafish optic tectum.
Figure 7: FESEM images of adult Drosophila brain ultrathin section mounted on Kapton tape.

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Acknowledgements

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.

Author information

Authors and Affiliations

Authors

Contributions

J.B. developed the staining concept, prepared the fish and Drosophila samples and prepared the manuscript. J.C.T., N.K. and R.S. imaged the Drosophila sections. R.S. assisted with the sectioning, imaging and overall block quality assessment. K.J.H., R.S., J.C.T. and N.K. improved ultrathin sectioning and collection. K.J.H. developed the method of collecting ultrathin sections on tape and built the ATUM (automatic tape-collecting ultra-microtome) devices used. J.W.L. helped motivate the effort to find better en bloc staining protocols, oversaw all the imaging experiments that were carried out in his laboratory and helped to interpret the image data. S.J.S. helped motivate the effort to improve en bloc staining, and oversaw and assisted with imaging experiments carried out at Stanford.

Corresponding author

Correspondence to JoAnn Buchanan.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

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)

Supplementary Fig. 2

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)

Supplementary Fig. 3

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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