https://doi.org/10.4081/ejh.2022.3364
How to stain nucleic acids and proteins in Miller spreads
Submitted: 30 November 2021
Accepted: 12 February 2022
Published: 25 February 2022
Accepted: 12 February 2022
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All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.
Authors
The spread technique proposed by Miller and Beatty in 1969 allowed for the first time the visualization at transmission electron microscopy of nucleic acids and chromatin in an isolated and distended conformation. The final step of staining the spread chromatin is of critical importance because it can strongly influence the interpretation of the results. We evaluated different staining techniques and the most part of them provided a good result. Specifically, well contrasted micrographs were obtained when staining with H3PW12O40 (PTA), as originally proposed by Miller and Beatty, and with two alternatives proposed here: uranyl acetate or terbium citrate staining. Quite good contrast of the spread DNA could be achieved also by using Osmium Ammine; while no or few contrast of nucleic acids was observed by staining with KMnO₄ and H3PMo12O40 (PMA) respectively.
Denker A, De Laat W. The second decade of 3C technologies: detailed insights into nuclear organization. Genes Dev 2016;30:1357-82.
DOI: https://doi.org/10.1101/gad.281964.116
Walter J, Joffe B, Bolzer A, Albiez H, Benedetti PA, Müller S. et al. Towards many colors in FISH on 3D-preserved interphase nuclei. Cytogenet Genome Res 2006;114:367-78.
DOI: https://doi.org/10.1159/000094227
Lakadamyali M, Cosma MP. Advanced microscopy methods for visualizing chromatin structure. FEBS Lett 2015;589:3023-30.
DOI: https://doi.org/10.1016/j.febslet.2015.04.012
Maiser A, Dillinger S, Längst G, Schermelleh L, Leonhardt H, Németh A. Super-resolution in situ analysis of active ribosomal DNA chromatin organization in the nucleolus. Sci Rep 2020;10:7462.
DOI: https://doi.org/10.1038/s41598-020-64589-x
Miller OL, Beatty BR. Visualization of nucleolar genes. Science 1969;164:955-7.
DOI: https://doi.org/10.1126/science.164.3882.955
Fakan S, Hernandez‐Verdun D. The nucleolus and the nucleolar organizer regions. Biol Cell 1986;56:189-205.
DOI: https://doi.org/10.1111/j.1768-322X.1986.tb00452.x
Mougey EB, O'Reilly M, Osheim Y, Miller Jr OL, Beyer A, Sollner-Webb B. The terminal balls characteristic of eukaryotic rRNA transcription units in chromatin spreads are rRNA processing complexes. Genes Dev 1993;7:1609-19.
DOI: https://doi.org/10.1101/gad.7.8.1609
Biggiogera M, Fakan S. Fine structural specific visualization of RNA on ultrathin sections. J Histochem Cytochem 1998;46:389-95.
DOI: https://doi.org/10.1177/002215549804600313
Fakan S, Leser G, Martin TE. Immunoelectron microscope visualization of nuclear ribonucleoprotein antigens within spread transcription complexes. J Cell Biol 1986;103:1153-7.
DOI: https://doi.org/10.1083/jcb.103.4.1153
Vázquez-Nin GH, Biggiogera M, Echeverría OM. Activation of osmium ammine by SO2-generating chemicals for EM Feulgen-type staining of DNA. Eur J Histochem 1995;39:101-6.
Sheridan WF, Barrnett, RJ. Cytochemical studies on chromosome ultrastructure. J Ultrastruct Res 1969;27:216-29.
DOI: https://doi.org/10.1016/S0022-5320(69)80013-4
Silverman L, Glick D. The reactivity and staining of tissue proteins with phosphotungstic acid. J Cell Biol 1969;40:761-7.
DOI: https://doi.org/10.1083/jcb.40.3.761
Höög L, Gluenz E, Vaughan S, Gull K. Ultrastructural investigation methods for Trypanosoma brucei. Methods Cell Biol 2010;96:175-96.
DOI: https://doi.org/10.1016/S0091-679X(10)96008-1
Hayat MA. Positive staining. Principles and techniques of electron microscopy (biological applications), 4th ed. Cambridge: Cambridge University Press; 2000. p. 242-366.
Hörer OL, Zaharia CN, Marcu A. Terbium fluorescence in aqueous solutions of nucleic acids. Rev Roum Biochim 1977;14:175-9.
Ringer DP, Howell BA, Kizer DE. Use of terbium fluorescence enhancement as a new probe for assessing the single-strand content of DNA. Anal Biochem 1980;103:337-42.
DOI: https://doi.org/10.1016/0003-2697(80)90620-X
Derenzini M, Farabegoli F. Selective staining of nucleic acids by osmium-ammine complex in thin sections from lowicryl-embedded samples. J Histochem Cytochem 1990;38:1495-501.
DOI: https://doi.org/10.1177/38.10.2205645
Biggiogera M, Courtens J, Derenzini M, Fakan S, Hernandez-Verdun D, Risueno MC et al. Osmium ammine: Review of current applications to visualize DNA in electron microscopy. Biol Cell 1996;87:121-32.
DOI: https://doi.org/10.1111/j.1768-322X.1996.tb00974.x
Luft JH. Permanganate; a new fixative for electron microscopy. J Biophys Biochem Cytol 1956;2:799-802.
DOI: https://doi.org/10.1083/jcb.2.6.799
Sutton JS. Potassium permanganate staining of ultrathin sections for electron microscopy. J Ultrastruct Res 1968;21:424-9.
DOI: https://doi.org/10.1016/S0022-5320(67)80150-3
Lawn AM. The use of potassium permanganate as an electron-dense stain for sections of tissue embedded in epoxy resin. J Biophys Biochem Cytol 1960;7:197-8.
DOI: https://doi.org/10.1083/jcb.7.1.197
Stockert JC, Blanco J, Ferrer J, Trigoso C, Tato A, Del Castillo P, et al. Tungsten and molybdenum heteropolyacids as staining and contrasting agents: Reactivity with epoxy resin-embedded cell and tissue structures. Acta Histochem 1989;2:151-8.
DOI: https://doi.org/10.1016/S0065-1281(89)80084-4
How to Cite
Zannino, L. ., & Biggiogera, M. (2022). How to stain nucleic acids and proteins in Miller spreads. European Journal of Histochemistry, 66(1). https://doi.org/10.4081/ejh.2022.3364
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