The spreading 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. This approach is beneficial since it can reveal many aspects of chromatin organization and function that otherwise can only be indirectly inferred by biochemical methods. The final step of staining chromatin spreads is critical because it can strongly influence the interpretation of the results. We evaluated different staining techniques, and almost all provided a good result. Specifically, well-contrasted micrographs were obtained when staining with H3PW12O40 (phosphotungstic acid, PTA), as originally proposed by Miller and Beatty, and with two alternatives proposed here: uranyl acetate or terbium citrate. Quite a good contrast of the spread DNA could also be achieved using osmium ammine; while no or little contrast of nucleic acids was observed by staining with KMnO4 (potassium permanganate) and H3PMo12O40 (phosphomolybdic acid, PMA) respectively.

How to stain nucleic acids and proteins in Miller spreads

Zannino, Lorena
;
Biggiogera, Marco
2022-01-01

Abstract

The spreading 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. This approach is beneficial since it can reveal many aspects of chromatin organization and function that otherwise can only be indirectly inferred by biochemical methods. The final step of staining chromatin spreads is critical because it can strongly influence the interpretation of the results. We evaluated different staining techniques, and almost all provided a good result. Specifically, well-contrasted micrographs were obtained when staining with H3PW12O40 (phosphotungstic acid, PTA), as originally proposed by Miller and Beatty, and with two alternatives proposed here: uranyl acetate or terbium citrate. Quite a good contrast of the spread DNA could also be achieved using osmium ammine; while no or little contrast of nucleic acids was observed by staining with KMnO4 (potassium permanganate) and H3PMo12O40 (phosphomolybdic acid, PMA) respectively.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11571/1472275
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