ARTICLE |
Correspondence to: Marco Biggiogera, Dipartimento di Biologia Animale, Laboratorio di Istologia, U. Of Pavia, Piazza Botta 10, 27100 Pavia, Italy.
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Summary |
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We describe a new technique that allows specific visualization of RNA at the electron microscopic level by means of terbium citrate. Under the conditions presented here, terbium binds selectively to RNA and stains nucleoli, interchromatin granules, peri-chromatin fibrils, perichromatin granules, and coiled bodies in the cell nucleus, whereas ribosomes are the only contrasted structures in the cytoplasm. All the cell components contrasted by terbium are known to contain RNA. When ultrathin sections are pretreated with RNase A or nuclease S1 (specific for single-stranded nucleic acids), staining does not occur. Neither DNase nor pronase influences the reaction. We conclude that terbium staining is selective for RNA and especially for single-stranded RNA. The staining can be performed on thin sections of material embedded both in epoxy and in acrylic resins. The technique is not influenced by the aldehyde fixative used and can also be utilized after immunolabeling. The endproduct is very fine and, although weak in contrast, is suitable for high-resolution observations. (J Histochem Cytochem 46:389395, 1998)
Key Words: electron microscopy, terbium, lanthanides, RNA staining, cytochemistry
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Introduction |
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The detection of nucleic acids plays an important role in ultrastructural cytochemistry (for reviews see
Thus far, the most widely used technique for detection of RNP-containing structures is the EDTA regressive stain of
With the aim of finding a simple technique for specific RNA detection in ultrathin sections, we have tested several members of the lanthanide family known to interact with nucleic acids. The purpose of the study was to find a specific, simple and reproducible technique that could be used on thin sections, independently of the fixation method (with the exception of osmium postfixation because of its electron density) or of the embedding resin used. Moreover, this technique should also allow the use of immunoprobes on the same section to provide additional data on other cell components.
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Materials and Methods |
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Mouse and rat liver, pancreas, and testes were fixed immediately after dissection in one of the following fixative solutions made in Sörensen phosphate buffer, pH 7.4: 1% or 2.5% glutaraldehyde (1 hr, 4C), 4% paraformaldehyde (2 hr, 4C), 2% paraformaldehyde with 0.2% glutaraldehyde (1 hr, 4C), 5% acrolein (1 hr, 4C). They were rinsed with buffer and free aldehyde groups were blocked by 0.5 M NH4Cl solution in PBS for 15 min on ice. Some specimens were dehydrated in ethanol and embedded in LR White resin; others were dehydrated in acetone and embedded in Epon. Finally, some specimens were dehydrated at progressively lower temperatures and embedded in Lowicryl K4M (
HeLa cells, infected with adenovirus type 5 (Ad5), fixed in 4% paraformaldehyde, and embedded in Lowicryl K4M, were kindly provided by Drs. F. PuvionDutilleul and E. Puvion (Villejuif, France).
Isolated salmon sperm DNA (Sigma; St Louis, MO) or isolated baker's yeast RNA (Sigma) was pre-embedded in 2% aqueous agarose, dehydrated, and embedded in LR White resin as additional specificity controls.
Thin sections (silver interference color) were cut and collected on grids, either uncoated or coated with a Formvarcarbon membrane. The reaction can be performed on copper, nickel, or gold grids.
Preparation of the Reagent
Terbium (III) nitrate pentahydrate 435 mg (Aldrich Chemicals; Milwaukee, WI) was dissolved in 5 ml of double-distilled or ultrapure water to obtain a 0.2 M solution. Then 5 ml of a 0.3 M solution of trisodium citrate 2-hydrate (Merck; Darmstadt, Germany) was prepared. Under continuous stirring, the terbium solution was added dropwise to the citrate and a white precipitate was formed. Then 1 N NaOH was added dropwise until the solution became transparent again and the precipitate was completely dissolved (this happens at pH 6.77.0). pH was adjusted to 8.28.5 with 1 N NaOH. At pH lower than 8.0, a precipitate is formed within 23 days. For this reason, the pH must be checked again after 24 hr and readjusted if necessary. The final solution of terbium citrate is stable for several weeks at room temperature (RT) and can be kept in a plastic syringe fitted with a 0.22-µm Millipore filter.
Staining Procedure for Epon Sections
Staining Procedure for Acrylic Sections
Controls
The following control reactions were carried out on para-formaldehyde-fixed, acrylate-embedded tissues:
To check the specific action of each enzyme, some grids were floated onto the corresponding buffer solution. The grids were then stained with terbium citrate as above.
Immunolabeling
Some grids were placed on 1% normal goat serum in PBS for 3 min and then incubated overnight at 4C on a mixture containing a monoclonal anti-DNA antibody (Progene; Heidelberg, Germany) diluted at 1:50 in PBS containing 0.05% Tween-20 and 0.1% bovine serum albumin. After washing with PBSTween and PBS, the grids were treated with normal goat serum as above and then incubated on goat anti-mouse IgM coupled to 15-nm gold particles (Aurion; Wageningen, The Netherlands). After drying, the grids were stained with terbium citrate as described.
EDTA Staining
Some of the sections were stained with the EDTA regressive technique, which preferentially reveals nuclear ribonucleo-proteins (
All grids were observed in a Philips CM12 electron microscope operating at 60 or 80 kV and equipped with a 30-µm objective aperture.
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Results |
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Treatment for 1 hr with a solution of alkaline sodium citrate does not add any significant contrast to the cell structures (Figure 1), and the result is comparable to unstained sections (not shown).
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After the EDTA regressive staining, the contrasted structures in an Epon section correspond to ribonucleoprotein (RNP) constituents. In the cytoplasm the ribosomes are stained and, in the nucleus, the nucleolus, perichromatin fibrils (PF), interchromatin granule (IG) clusters, perichromatin granules (PG), and coiled bodies represent the only contrasted structural constituents. Condensed chromatin is grayish (Figure 2) or bleached, depending on the incubation time with EDTA.
When the Epon sections are first treated with Na-citrate and then with terbium citrate, only structures known to contain RNA are contrasted. In the nucleus, the nucleolus, PF, and IG and PG are contrasted (Figure 3). In the cytoplasm, only ribosomes are stained (Figure 3, inset).
After anti-DNA immunolabeling on acrylic sections of liver cells followed by terbium staining, the gold grains indicating DNA distribution do not superimpose on contrasted structures, such as PF and IG (Figure 4).
Pretreatment of the thin sections with RNase completely abolishes the staining both in the nucleus and in the cytoplasm (Figure 5). A similar result is obtained when digestion with nuclease S1 (which removes single-stranded nucleic acids) and terbium staining are performed (Figure 6). It is interesting that when S1 nuclease is used the contrast of chromatin is enhanced, regardless of whether or not Tb staining was performed. A similar phenomenon has previously been reported when RNase digestion was applied to ultrathin sections (Yotsuyanagy 1965). It therefore appears that the nuclease can bind to chromatin, possibly because of its association either with DNA or chromatin proteins, thus increasing the mass of the chromatin-containing area.
DNase pretreatment of the sections does not affect the RNA staining, as shown in Figure 7. In addition, specific staining is also present after pronase treatment. Most of the proteins are removed (especially from condensed chromatin areas), but the nuclear and cytoplasmic RNA remains contrasted (Figure 8).
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Additional controls were performed by staining thin sections of isolated DNA or RNA. RNA molecules are contrasted by terbium, whereas DNA is not stained (results not shown).
As a further control, HeLa cells infected for 17 hr with Ad 5 virus were used. At this stage of infection, Ad 5 is present in capsids and also forms areas of ssDNA and zones containing only viral RNA (
As for the embedding media and fixation procedures, good results are obtained with formaldehydeglutaraldehyde fixation and embedding in Lowicryl K4M (not shown) or LR White (Figure 10) resin, although acrylic resins show a rather high inherent contrast in ultrathin sections. Epon sections give good results in terms of staining, and chromatin is clearly devoid of contrast (Figure 11).
Paraformaldehyde or acrolein fixation gives rise to results comparable to those of glutaraldehyde in all the resins tested (not shown).
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Discussion |
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Our results indicate that the terbium citrate technique gives rise to selective staining of RNA in ultrathin sections at the electron microscopic level. The staining is abolished by previous treatment with RNase or nuclease S1 but not with DNase or pronase. Because staining is abolished by single-stranded RNA digestion and Tb citrate does not react with ssDNA in adenovirus 5-infected HeLa cells, terbium obviously stains only ssRNA. The technique can be applied to Epon and acrylate sections, is not influenced by the different aldehyde fixatives, and can also be performed after immunolabeling. Finally, the endproduct, although weak in contrast, is very fine and allows high-resolution visualization.
We present here a new technique for specific staining of RNA in ultrathin sections. This method is rapid (involving only two steps for Epon sections or one for acrylic sections), can be performed independently of the fixation (with the exception of osmium, which must be avoided because of its electron density and its ability to bind also to nucleic acids) and embedding media. Most importantly, Tb staining can be performed on tissue sections after embedding, thus avoiding the problem of having tissue blocks "committed"solely to one staining purpose. In addition, the Tb method can successfully be used after immunocytochemistry, thus offering application on specimens prepared routinely for purposes of general investigation.
Our control experiments confirmed that the stained material is indeed RNA. RNase or nuclease S1 digestion prevents staining, whereas DNase or pronase has no effect. This further indicates that the resulting electron-dense product is due to RNA, without any interference by DNA or proteins, and that, in addition, Tb appears to bind to single-stranded RNA, which represents the major part of the RNA in a cell. This is also confirmed by staining of Ad 5-infected HeLa cells, in which Tb does not react with the areas containing viral ssDNA formed during the infection.
The resolution of the method (which obviously depends on the fineness of the endproduct) is very good and allows high-magnification studies. In some cases, a single stained filament of RNA can be seen within perichromatin granules (inset in Figure 10). We must stress the fact that the final contrast under the microscope is weak and is roughly comparable to that obtained with the results of DNA staining with osmium ammine on epoxy sections (
As for the reaction mechanism, it is not clear how terbium (III) ions bind only to RNA on thin sections. The structural and chemical differences between DNA and RNA are few. These differences obviously account for the specific staining of DNA with the Feulgen-type technique. Thus far there has been no specific staining method for RNA in ultrastructural cytochemistry. Available data suggest that Tb ions in their oxidation form 3+ can interact with single-stranded nucleic acids (
Other rare earth elements, such as europium and ytterbium (and also lutetium, the heaviest of the rare earths), share the same properties, but they form more abundant and coarser precipitate deposits on the sections (unpublished observations). Several terbium salts were tested and only the citrate (which is stable at alkaline pH) gives rise to good and reproducible results.
The staining time with Tb is not critical: the same results can be obtained from 1 to 18 hr of incubation. The first staining step (treatment with alkaline sodium citrate, used here only for Epon sections) is necessary to shorten the staining time. If the solution of terbium citrate is used alone, an overnight incubation is necessary. The fact that the staining pattern does not change between 1 and 18 hr indicates that the staining reaches a saturation point, thus preventing overstaining (and unspecific staining).
Different applications of this staining technique are now in progress, including experiments aiming to follow RNA distribution after Tb staining by use of electron spectroscopic imaging. Moreover, because Tb ions are photoluminescent, the possibility of using this method in fluorescence microscopy, and perhaps in flow cytometry, is under investigation.
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Acknowledgments |
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Supported by the Swiss National Science Foundation (31-37432.93 and 31-43333.95).
We thank Drs M. Malatesta (Urbino, Italy), A. Fraschini (Pavia, Italy), G.H. VázquezNin, and O.M. Echeverría (Mexico City) for testing the technique in their laboratories. Thanks are due to Drs F. PuvionDutilleul and E. Puvion (Villejuif, France) for kindly providing a specimen of HeLa cells infected with Ad 5. Drs S. Pissavini and P. Froidvaux are acknowledged for discussion on the chemistry of rare earths. The technical assistance of Ms J. Fakan, V. Mamin, F. Flach Biggiogera, N. Ruchonnet, and P. Veneroni is gratefully acknowledged.
Received for publication June 16, 1997; accepted October 13, 1997.
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