Journal of Histochemistry and Cytochemistry, Vol. 46, 389-396, Copyright © 1998, The Histochemical Society, Inc.


ARTICLE

Fine Structural Specific Visualization of RNA on Ultrathin Sections

Marco Biggiogeraa,b and Stanislav Fakana
a Centre of Electron Microscopy, University of Lausanne, Lausanne, Switzerland, Dipartimento di Biologia Animale, Laboratorio di Istologia
b Centro di Studio per l'Istochimica del CNR, University of Pavia, Pavia, Italy

Correspondence to: Marco Biggiogera, Dipartimento di Biologia Animale, Laboratorio di Istologia, U. Of Pavia, Piazza Botta 10, 27100 Pavia, Italy.


  Summary
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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 46:389–395, 1998)

Key Words: electron microscopy, terbium, lanthanides, RNA staining, cytochemistry


  Introduction
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

The detection of nucleic acids plays an important role in ultrastructural cytochemistry (for reviews see Moyne 1980 ; Hayat 1993 ). Although a specific, Feulgen-type method for DNA staining has existed since 1973 (Cogliati and Gautier 1973 ), thus far there has been no selective staining method for RNA on thin sections. The use of platinum quinoline has been proposed by Scott 1973 but requires application on cryostat sections, which must then be embedded in resin after the reaction, thus seriously limiting both the preservation of the structure and the use of existing specimens prepared in a standard way. Several techniques are available for the detection of both DNA and RNA, involving the application of an acriflavine–PTA complex (Chan-Curtis et al. 1970 ), ethidium bromide–PTA (Biggiogera and Flach Biggiogera 1989 ), or osmium ammine without acid hydrolysis (Derenzini and Farabegoli 1990 ). However, all of these methods require an enzymatic removal of DNA from the sections in order to detect only RNA. In addition, different techniques, such as RNase–gold complexes (Bendayan 1982 ), anti-RNA antibodies, or incorporation of labeled precursors have been used for specific visualization of RNA.

Thus far, the most widely used technique for detection of RNP-containing structures is the EDTA regressive stain of Bernhard 1969 . However, this method, based on the chelation of uranyl ions by neutral EDTA, is preferential, and stains nuclear ribonucleoprotein structural constituents regardless of whether or not they contain RNA (Bernhard 1969 ). Recently a renewed interest has characterized several studies involving the use of elements of the lanthanide family, such as europium and terbium. Data in the literature point out that these ions in their oxidation form 3+ can interact with single-stranded nucleic acids (Horer et al. 1977 ) at their guanine sites (Ringer et al. 1978 ), at the phosphate in unpaired residues (Topal and Fresco 1980 ), and especially with guanosine monophosphate in RNA (Ringer et al. 1980 ). At the electron microscopic (EM) level, these ions (particularly lanthanum) have been known to precipitate in the presence of phosphate groups (Hayat 1993 ), and members of the family, such as cerium, are now routinely used for EM detection of phosphatase activity (Van Noorden and Frederiks 1993 ).

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.


  Materials and Methods
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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 (Carlemalm et al. 1982 ).

HeLa cells, infected with adenovirus type 5 (Ad5), fixed in 4% paraformaldehyde, and embedded in Lowicryl K4M, were kindly provided by Drs. F. Puvion–Dutilleul 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 Formvar–carbon 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 6.7–7.0). pH was adjusted to 8.2–8.5 with 1 N NaOH. At pH lower than 8.0, a precipitate is formed within 2–3 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

  1. Incubate the grids on drops of a 0.5 M solution of sodium citrate brought to pH 12 with 1 N NaOH (this solution is also very stable) for 1 hr at RT.

  2. Rapidly blot the grid with filter paper to remove excess solution.

  3. Stain on terbium citrate for 1 hr at RT.

  4. Rinse with distilled water for 15–30 sec, blot, and dry.

Staining Procedure for Acrylic Sections

  1. Stain on terbium citrate diluted 1:5 with distilled water for 1 hr at 37C.

  2. Rapidly blot the grid with filter paper to remove excess solution.

  3. Put the grid on a large drop of water for 10–15 sec.

  4. Blot and dry.

Controls
The following control reactions were carried out on para-formaldehyde-fixed, acrylate-embedded tissues:

  1. RNase A (Sigma) treatment was performed with 0.1% RNase solution in triethanolamine buffer (pH 7.0) for 5 hr at 37C.

  2. DNase I (Sigma) was used at 0.1% concentration in phosphate buffer containing 2 mM MgCl2, for 5 hr at 37C.

  3. Nuclease S1 from Aspergillus oryzae (Sigma), specific for single-stranded nucleic acids, was used at a concentration of 100 U/ml in acetate buffer (pH 4.5) containing 50 mM NaCl and 3 mM ZnCl2, for 5 hr at 37C.

  4. Pronase (Sigma) 0.1% in water for 15–30 min at RT.

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 PBS–Tween 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 (Bernhard 1969 ).

All grids were observed in a Philips CM12 electron microscope operating at 60 or 80 kV and equipped with a 30-µm objective aperture.


  Results
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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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Figure 1. Mouse liver, glutaraldehyde fixation, LR White embedding. The section has been treated with sodium citrate alone. No contrast is present after the first part of the reaction, and the cell structures are difficult to interpret both in the nucleus (n) and in the cytoplasm (c). Bar = 0.1 µm.

Figure 2. Rat hepatocyte, glutaraldehyde fixation, Lowicryl K4M embedding. EDTA regressive staining. Nucleolus, PF (large arrow), and PG (small arrow) are contrasted, whereas condensed chromatin appears bleached. Bar = 0.5 µm.

Figure 3. Rat hepatocyte, glutaraldehyde, Epon. Terbium staining. The nucleolus, IG (arrowhead), PF (large arrow), and PG (small arrow) are the only nuclear structures stained by Tb. Chromatin (c) remains unstained. (Inset) In the cytoplasm, the ribosomes are clearly stained. Bars = 0.5 µm.

Figure 4. Mouse Kuppfer cell, paraformaldehyde, LR White, After anti-DNA immunolabeling and Tb staining, contrasted structures are present at the periphery of the areas labeled by the gold grains. Bars = 0.5 µm.

Figure 5. Mouse hepatocyte, paraformaldehyde, LR White. When a pretreatment with RNase is performed on thin sections, Tb does not bind and the overall contrast of the cell is comparable to that of Figure 1. n, nucleus; c, cytoplasm. Bar = 0.5 µm.

Figure 6. Mouse hepatocyte, paraformadehyde, LR White. After digestion with nuclease S1 before Tb staining, RNA-containing structures are not contrasted. n, nucleus; c, cytoplasm. Bar = 0.5 µm.

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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Figure 7. Mouse hepatocyte, paraformaldehyde, LR White. Pretreatment with DNase does not hinder the staining of RNA by Tb. PF (arrow) and IG (arrowhead) are easily detectable. Ribosomes are also stained. Bar = 0.5 µm.

Figure 8. Mouse Kuppfer cell, paraformaldehyde, LR White. Pronase treatment before Tb staining. Removal of proteins provides the same staining patterns as in Figure 7. Note the massive extraction from the condensed chromatin (c). Bar = 0.5 µm.

Figure 9. HeLa cell after 17-hr infection with Ad 5, paraformaldehyde, Lowicryl K4M. Anti-DNA labeling and Tb staining. Tb stains the dense areas corresponding to the viral RNA ring (arrowheads) but not the ssDNA area, which is labeled by the anti-DNA probe. Bar = 0.5 µm.

Figure 10. Rat hepatocyte, glutaraldehyde, LR White embedding, Tb staining. A coiled body (large arrow), PF (small arrow), a cluster of IGs (asterisk), and PGs (arrowheads) are shown. (Inset) A single PG with a distinct stained fibrillar structure inside. Bars = 0.1 µm.

Figure 11. Rat hepatocyte, paraformaldehyde, Epon, Tb staining. Condensed chromatin (c) is unstained; PF and ribosomes are contrasted. Note the presence of stained filaments inside the nuclear pore (arrow). Bar = 0.1 µm.

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 (Puvion-Dutilleul et al. 1992 ). In Figure 9, a HeLa cell nucleus is shown after anti-DNA labeling and Tb staining. Only the areas containing the viral RNA appear to be stained. The zones of ssDNA are not contrasted by Tb but are labeled by the anti-DNA antibody.

As for the embedding media and fixation procedures, good results are obtained with formaldehyde–glutaraldehyde 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).


  Discussion
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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 (Cogliati and Gautier 1973 ). During direct observation in the electron microscope, the contrasted structures become readily visible after a few minutes of adaptation and the unstained chromatin areas (which are white) are of great help in localizing the cells. It must be noted that, for acrylic-embedded tissues, the sections should be cut at a white–silver interference color thickness, because acrylic resins confer a higher inherent contrast to cell structures. Reducing the section thickness is therefore a simple way of increasing the signal-to-noise ratio. Nevertheless, the contrast on the negatives and on printed micrographs is satisfactory. Any attempt to increase the contrast by use of additional reagents has failed; even a 5-sec poststaining with lead citrate increases the contrast but causes a definite loss of specificity, because Tb also binds to proteins.

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 (Horer et al. 1977 ) at their guanine sites (Ringer et al. 1978 ), at the phosphate in unpaired residues (Topal and Fresco 1980 ), and particularly with guanosine monophosphate in RNA (Ringer et al. 1980 ).

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.


  Acknowledgments

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. Vázquez–Nin, and O.M. Echeverría (Mexico City) for testing the technique in their laboratories. Thanks are due to Drs F. Puvion–Dutilleul 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.


  Literature Cited
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Bendayan M (1982) Ultrastructural localization of nucleic acids by the use of enzyme-gold complexes: influence of fixation and embedding. Biol Cell 43:153-156

Bernhard W (1969) A new staining procedure for electron microscopical cytology. J Ultrastruct Res 27:250-265[Medline]

Biggiogera M, Flach Biggiogera F (1989) Ethidium bromide– and propidium iodine–PTA staining of nucleic acids at the electron microscopic level. J Histochem Cytochem 37:1161-1166[Abstract]

Carlemalm E, Garavito RM, Villiger W (1982) Resin development for electron microscopy and an analysis of embedding at low temperature. J Microsc 126:123-143

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Cogliati R, Gautier A (1973) Mise en évidence de l'ADN et des polysaccharides á l'aide d'un nouveau réactif "de type Schiff.". CR Acad Sci 276:3041-3044

Derenzini M, Farabegoli F (1990) Selective staining of nucleic acids by osmium ammine complex in thin sections from Lowicryl-embedded samples. J Histochem Cytochem 28:1495-1501

Hayat H (1993) Stains and Cytochemical Methods. New York, London, Plenum Press

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Yotsuyanagi Y (1965) Mise en évidence au microscope électronique d'ADN dans le mitochondries de la levure; sa signification possible dans l'hérédité cytoplasmique de la déficience respiratoire. J Microsc 4:170-182