Does any one know a good reagent (or protocol) to stain nucleic acids (RNA) for electron microscopy (either negative stain or positive stain)?
Dear Pengfei Ding,
I have used a special EM stain,long ago,to stain nucleic acids differentially with regard to their DNA/RNA composition by Bismuth staining. It is not widely known, but I will try to provide you with some links. Kind regards, Harrie Verhoeven.
Uranyl Acetate (UA) is the best staining material for nucleic acids. The advantage of UA is that produces the highest electron density and image contrast as well as imparting a fine grain to the image due to the atomic weight of 238 of uranium. The uranyl ions bind to proteins and lipids with sialic acid carboxyl groups such as glycoproteins and ganglioside and to nucleic acid phosphate groups of DNA and RNA.
The attachment will guide you to prepare a staining solution.
While my cherished colleagues above posted good solutions (and naturally UO2-acetate is the most important candidate for nucleic acids (mainly for DNA) in double staining, also in its other chemical forms often applied to ultrathin sections, namely uranyl-formate or uranyl magnesium acetate (7,5 % for instance), I would like to add at least also the link to another RG-Question asked & answered multiplicatively 3 years ago: cf.:
https://www.researchgate.net/post/What_is_the_last_method_for_staining_of_DNA_and_DNA_detection_in_ultra_structure_samples_for_Electron_Microscopy_EM .
Michael J. Dykstra, Laura E. Reuss - 2003 in their book: Biological Electron Microscopy: Theory, Techniques, and Troubleshooting describe ".....However, RNA is more reactive with lead stains"...... @cf: https://books.google.at/books?isbn=0306477491".
Further candidates:
(phosphotungstic acid, phosphomolybdic acid), "EDTA-regressive" staining for ribonucleoproteins results in chromatin bleaching and stains RNA-containing Structures (Bernhard W,1969, A new staining procedure for electron microscopical cytology. J. Ultrastruct.Res.27,250-265, ARTICLE available only via PPV=Pay Per View: cf: http://www.sciencedirect.com/science/article/pii/S002253206980016X ).
Ethidium bromide- and propidium iodide-PTA staining for the demonstration of RNA-containing structures (Biggiogera and Flach-Biggiogera 1989, Ethidium bromide- and propidium iodide-PTA staining of nucleic acids at the electron microscopy level. J.Histochem.Cytochem 37, 1161-1166. Abstract: http://journals.sagepub.com/doi/abs/10.1177/37.7.2471727, PDF @: http://journals.sagepub.com/doi/pdf/10.1177/37.7.2471727
Bromination of (LR-White) ultrathin sections (mounted on nickel grids) followed by specific antibodies against bromo-deoxyuridine might be another way: cf:
CALABUIG et al, Specific detection of RNA on ultra-thin sections, Journal of Structural Biology 152 (2005) 146–148:
Abstract (only for convenience)
In this paper, we describe a specific method for ultrastructural detection of RNA. Our method is based in the bromination ‘‘in situ’’ of uridine residues in the RNA, which allows the detection of brominated RNA with specific antibodies against bromo-deoxyuridine. With this method we can achieve high specificity and resolution, and it can be applied to Epon or acrylic resin embedded material.
(C) 2005 Published by Elsevier Inc. Article Abstract cf.: http://www.sciencedirect.com/science/article/pii/S1047847705001425 ; PDF available only via PPV.
Keywords: Electron microscopy; Nucleic acid detection; RNA
EM-Staining for DNA and RNA have been reviewed (only one example out of some other references) by LEWIS PR and KNIGHT DP 1977: Staining Methods for sectioned material. In series: GLAUERT A.M.(Ed), Practical Methods in Electron Microscopy, Vol.5, Part I, pp. 1-311, Elsevier / North Holland Biomedical Press. Amsterdam (NL).
It might be worth thinking about the influence of (different) fixatives usually applied to tissue(s) for analytical TEM(positive stain/contrast )
Best wishes and good luck!
NB: This answer was edited for /with the links to mentioned articles in respective journals 50 min afetr initial posting.
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