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High-pressure treatment of polytene chromosomes improves structural resolution

Nature Methods volume 4, pages 483–485 (2007)Cite this article

Abstract

The exceptional cytology provided by polytene chromosomes has made Drosophila melanogaster a premier model for chromosome studies, but full exploitation of polytene cytology is impeded by the difficulty in preparing high-quality chromosome spreads. Here we describe use of high pressure to produce formaldehyde-fixed chromosome spreads, which upon light-microscopy examination reveal structural detail previously observed only in electron microscopy preparations. We demonstrate applications to immunofluorescence and in situ hybridization.

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Figure 1: Comparison of spreads of D. melanogaster polytene chromosomes prepared using the traditional and new techniques.
Figure 2: Improved imaging of the hsp70 87A and 87B heat-shock puffs.
Figure 3: Improved cytological resolution of immunostained samples using the new protocol, as demonstrated by anti–lac repressor staining.

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Acknowledgements

Development of the method was supported by the US National Institutes of Health grant 2RO1GM058460 to A.S.B.

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Authors and Affiliations

  1. Department of Cell and Developmental Biology, University of Illinois, Urbana-Champaign, B107 Chemical and Life Sciences Building, 601 S. Goodwin Ave., Urbana, 61801, Illinois, USA

    Dmitri V Novikov, Igor Kireev & Andrew S Belmont

  2. Department of Electron Microscopy, A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, 119899, Russia

    Igor Kireev

Authors
  1. Dmitri V Novikov
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  2. Igor Kireev
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  3. Andrew S Belmont
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Contributions

D.V.N. improved the polytene chromosome spreading technique, developed the high-pressure treatment approach and performed the light microscopy; I.K. optimized and performed the electron microscopy sample preparation and immunostaining; A.S.B. coordinated the project and provided general guidance for the work.

Corresponding author

Correspondence to Andrew S Belmont.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Mechanical equipment and materials. (PDF 203 kb)

Supplementary Fig. 2

Average quality spreads of D. melanogaster salivary gland polytene chromosomes, prepared using the high pressure method. (PDF 163 kb)

Supplementary Fig. 3

Comparison of chromosomal spreads at the same location prepared by different techniques: 87A-C heat-shock puffs in polytene chromosomes of D. melanogaster salivary glands. (PDF 124 kb)

Supplementary Fig. 4

Boundary element scs' maps to junction between decondensed region within 87A heat shock puff and minor bands A8-10. (PDF 854 kb)

Supplementary Fig. 5

Comparison of light microscopy (described protocol) with EM. (PDF 1004 kb)

Supplementary Fig. 6

Comparison of the resolution in experiments with immunostaining obtained using conventional procedures with images acquired as described here. (PDF 902 kb)

Supplementary Video 1

Spreading and pressing procedures. (WMV 2615 kb)

Supplementary Methods (PDF 41 kb)

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Novikov, D., Kireev, I. & Belmont, A. High-pressure treatment of polytene chromosomes improves structural resolution. Nat Methods 4, 483–485 (2007). https://doi.org/10.1038/nmeth1049

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