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Increasing evidence demonstrates that RNA nucleotides undergo epigenetic modifications, such as methylation on cytosine. Although the presence of modified bases on mRNA has been proven, their molecular significance is largely undefined. We describe here a methodology to dissect the timing of modification of cytosine to 5-methylcytosine (5mC or m 5 C) in relation to RNA elongation and processing. To do this we use chlorouridine and iodouridine, two synthetically modified nucleotide bases which can be recognized by RNA polymerase II and incorporated into nascent RNA. These modified bases are added to a cell culture for defined intervals of time, and then immunocytochemical staining using antibodies against the modified nucleotides is carried out. This procedure allows us to identify the range of time in which 5mC is produced in nascent mRNA. This method provides the ultra-resolution of electron microscopy and allows following nascent RNA molecules during their elongation.

5-Methylcytosine is the major epigenetic modification occurring on DNA. It is known to be involved not only in gene expression regulation but also in the control of chromatin structure. However, this modification is also found on different types of RNA, including mRNA. Generally, biomolecular techniques are applied for studying the epigenetic profile of nucleic acids. Here, we describe the ultrastructural detection of 5-methylcytosine as an unusual approach to localize this modification on chromatin regions and/or RNA single molecules. This tool requires a careful sample preparation to preserve antigen epitopes that will be revealed immunocytochemically by a specific anti-5-methylcytosine antibody. The multiple staining procedures that can be adopted allow the identification of both DNA or RNA. A semiquantitative analysis can also be carried out.

The osmium ammine staining allows the specific detection of DNA in the cell nucleus and represents one of the most used techniques for EM cytochemistry.The procedure is a Feulgen-type reaction, consisting of an acid hydrolysis to obtain free aldehyde groups on DNA followed by their binding to osmium ammine, a Schiff-type reagent. Osmium ammine is polyamminic electron-dense compound commercially available.Here, we describe the staining procedure for ultrathin sections and the different procedures for the preparation of the reagent for acrylic and epoxy sections.

Although the EDTA regressive technique allows the visualization of RNPs, this widely used method is not intended to be specific for RNA alone. A fine ultrastructural visualization of RNA on ultrathin sections can be obtained with terbium citrate: this method gives a weak contrast but a very fine end product which allows observations at a high resolution level.The procedure is very simple since it consists only of a period of incubation and very short washes, which are the crucial point of this technique to avoid the Tb removal from RNA. This method does not require any special type of fixation and embedding.

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.

Annexin V is a Ca(++)-binding protein which is widely used as a marker for apoptotic cells, as it binds to the phosphatidylserine residues exposed at the surface of apoptotic cells. In this paper, we describe a method for the immunogold-labeling of biotin-conjugated Annexin V, to detect apoptotic thymocytes at electron microscopy. Etoposide-treated thymocytes were reacted in tissue culture medium with biotin-conjugated Annexin V, fixed with glutaraldehyde, and processed for resin embedding; thin sections were incubated with antibiotin antibodies coupled with colloidal gold. Cytometric estimates of the apoptotic index were also performed by evaluating either the DNA content after propidium iodide staining or the light-scattering values, as well as the positivity for fluorescein isothiocyanate-conjugated anti-biotin antibodies. At electron microscopy, gold-labeling of Annexin V was located on the plasma membrane only of apoptotic thymocytes and on cytoplasmic debris, likely resulting from the typical apoptotic blebbing. Unlabeled thymocytes always showed normal, non-apoptotic nuclear morphology. The application of Annexin V labeling at electron microscopy will allow a more refined description of the morphological events occurring during apoptosis.

This review has been collectively written. The contribution of the authors is mentioned for each part. References have been grouped at the end of the review. The objective of this review is to outline the principle of the method for electron microscopy, to emphasize the major applications and recent developments of this technique for DNA detection and finally to compare this technique with some other methods of DNA detection.

We present herein an improved method for the use of osmium ammine in a Feulgen-type reaction for specific staining of DNA at EM level and an analysis of its Schiff-type reagent behaviour. The activation of osmium ammine to a Schiff-type reagent (so far routinely performed by bubbling with gaseous SO2) can be accomplished by adding S0(2)-generating chemicals as for light microscopy. When used after HCI hydrolysis on epon or acrylate sections, activated osmium ammine behaves like a Schiff-type reagent, and the DNA staining can be selectively and completely abolished by aldehyde blocking agents. This preparation has the advantage of eliminating the use of gaseous SO2 thus rendering the technique more widely available to laboratories which cannot handle gas cylinders containing SO2. We recommend the use of osmium ammine in 8N acetic acid and 40mM sodium metabisulfite for 1 h at 37 degrees C for epon sections, and in 0.2N HCl and 0.2M metabisulfite for 30 min at room temperature for acrylate sections.

Ultra-thin sections of various tissues were stained with ethidium bromide or propidium iodide, two fluorescent markers widely used for quantitation of nucleic acids. The fluorochromes, tested at different concentrations, were then revealed by incubation of the sections with neutralized phosphotungstic acid. We showed that at the electron microscopic level only nucleic acid-containing structures are revealed. Chromatin, nucleolus, and ribosomes appear to be stained by the end-product of the reaction. Furthermore, controls with proteases and nucleases showed that the staining is related to the binding of the fluorochromes to DNA and RNA and to the subsequent detection of the dyes by neutralized PTA.