How to stain nucleic acids and proteins in Miller spreads

Abstract

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.

Figure 1.

The micrographs show chromatin spread from Hela cell lysates, stained by different techniques. a) PTA staining. This staining, traditionally used, binds to both nucleic acids (arrows) and proteins (arrowheads), which appear well contrasted. b) Uranyl acetate staining. It is possible to appreciate well-defined and contrasted nucleic acids (arrows) with some associated proteins (arrowheads). The image is free from precipitates. Example of immunocytochemistry on chromatin spread: protein complexes labeled with 12 nm gold grain conjugated with a secondary antibody indicating the presence of P1/P2 proteins, components of large ribosome subunit (white arrowheads). c) Terbium citrate staining. Terbium provides a marked contrast to nucleic acids (arrows) and proteins (arrowheads). Areas delimited by the squares are reported on the right of each image at higher magnification to show in detail the contrast provided to the spread chromatin by each staining technique. Scale bars: 1 μm.

Figure 2.

a) Osmium ammine staining. Feebly contrasted nucleic acids can be appreciated (arrow), but evident precipitates appear (asterisks). Proteins are not contrasted. b) Staining with Osmium Ammine preceded by acid hydrolysis (HCl 0.1N). As in d, the acid hydrolysis additional step does not improve the staining result. Note the weakly contrasted nucleic acids (arrows). Bars: 1 μm. a.,b. Areas delimited by the squares are reported on the right of each image at higher magnification to show in detail the contrast provided to the spread chromatin by each staining technique.

Figure 3.

The micrographs show spread cell lysate from Hela cells. Examples of inefficient contrast attempts. a) KMnO₄ staining. The image appears free from precipitates, but no contrast of nucleic acids can be appreciated; only protein components are visible (arrowheads). b) PMA staining. The image shows large precipitates (asterisks), which severely disturbs the observation of the feebly contrasted nucleic acids (arrows). Areas delimited by the squares are reported on the right of each image at higher magnification to show in detail the contrast provided to the spread chromatin by each staining technique. Scale bars: 1 μm.