The Feulgen reaction 75 years on

 The Feulgen reaction proposed by Feulgen and Rossenbeck 75 years ago is one of the cytohistochemical reactions most widely used in biology and medicine. It allows DNA in situ to be specifically stained based on the reaction of Schiff or Schiff-like reagents with aldehyde groups engendered in the deoxyribose molecules by HCl hydrolysis. The staining intensity is proportional to the DNA concentration. Current applications of the Feulgen reaction are mainly concerned with DNA quantification in cell nuclei by image cytometry for ploidy evaluation in tumor pathology. From the morphological point of view, specific demonstration of DNA in cell structures at the light microscopic level is very little used nowadays. On the other hand, application of the Feulgen principles to electron microscopy have recently allowed specific DNA-staining procedures to be developed for the study of the structural organization of DNA in situ. Accepted: 13 January 1999     

Modeling heterochromatin regions in transgenic mice

Transgenic mice carrying bovine satellite DNA IV were obtained. The size of the transgene integrated into the mouse genome was approximately 390 kb (about 100 transgene copies) as determined by a semiquantitative PCR. Restriction analysis with isoschizomeric restrictases HpaII and MspI, showed that the alien DNA was methylated. In the genome of a transgenic founder male, two integration sites for satellite DNA IV were revealed by in situ hybridization and in situ PCR. These sites are situated on two different chromosomes: in pericentromeric heterochromatin and within a chromosomal arm. In transgenic mice, de novo formation of heterochromatin regions (C-block and the CMA3 disk within the centromeric heterochromatin of another chromosome) was revealed by C-banding and staining with chromomycin A3. This formation is not characteristic of mice, because their chromosomes normally contain no interstitial C-blocks or sequences intensely stained by chromomycin A3.     

Use of non-radioactive DNA probes for the characterization of adult T-cell leukemia cells

DNA fragments were labeled with dinitrophenyl (DNP) residues by the reaction with 2,4-dinitrobenzaldehyde in alkaline condition and the labeled DNA was used as a probe for non-radioactive in situ hybridization. DNP-labeled DNA probes for T cell receptor beta chain, c-myc and HTLV-1 were hybridized in situ to mRNA on cell specimens fixed with Carnoy's fixative. DNA-mRNA hybrids were detected immunohistochemically using anti-DNP antibodies. Cytoplasms of adult T cell leukemia cells were stained with varied intensity when these probes were used. More than 70% of cells were positively stained with T cell receptor probe. However, less than 30% of cells were stained with c-myc and HTLV-1 probes. The present study indicates that non-radioactive in situ hybridization can be used for the characterization and classification of leukemia.     

Detection of Trypanosoma cruzi DNA within murine cardiac tissue sections by in situ polymerase chain reaction

The use of in situ techniques to detect DNA and RNA sequences has proven to be an invaluable technique with paraffin-embedded tissue. Advances in non-radioactive detection systems have further made these procedures shorter and safer. We report the detection of Trypanosoma cruzi, the causative agent of Chagas disease, via indirect and direct in situ polymerace chain reaction within paraffin-embedded murine cardiac tissue sections. The presence of three T. cruzi specific DNA sequences were evaluated: a 122 base pair (bp) sequence localized within the minicircle network, a 188 bp satellite nuclear repetitive sequence and a 177 bp sequence that codes for a flagellar protein. In situ hybridization alone was sensitive enough to detect all three T. cruzi specific DNA sequences.     

DNA cytofluorometry in micro-dissected paraffin embedded breast tissue: description of a method

DNA microfluorometry on smears obtained from paraffin embedded tissue has been shown to be a distinctive possibility. In this paper a simple method for the detachment of cells from mammary ducts and ductules is described. Areas of interest were selected in conventional slides stained with Haematoxylin and Eosin. The corresponding paraffin embedded block was then dewaxed. The areas under study were retraced with a stereomicroscope and the cells within ducts and ductules were scraped out with a 0.4 mm diameter fine needle. Cells were isolated with mechanical and enzymatic procedures and stained with the Feulgen reaction. The DNA content of single cells was then measured using a microfluorometer.     

Human serum deoxyribonuclease assay in (3H)DNA-polyacrylamide gels without staining artifacts

A method is described for detecting and evaluating serum deoxyribonuclease (DNase) activities after their separation by disc electrophoresis in polyacrylamide gels containing radioactively labeled DNA.After the electrophoretic run the gels are sliced, incubated in an appropriate buffer, and the amount of diffusible radioactive DNA fragments formed by the action of DNases in the incubation buffer is determined.The method has a high sensitivity as well as a quantitative reproducibility within ±5% even at low enzyme activities down to 10 pg Worthington DNase I equivalents.This method has been found superior to procedures that use staining of the gels for unhydrolyzed DNA, where irrelevant stained bands may invalidate the results. Thus, we get meaningful results even with human serum.     

Three-dimensional electron microscopy of ribosomal chromatin in two higher plants: a cytochemical, immunocytochemical, and in situ hybridization approach

We report the 3-D arrangement of DNA within the nucleolar subcomponents from two evolutionary distant higher plants, Zea mays and Sinapis alba. These species are particularly convenient to study the spatial organization of plant intranucleolar DNA, since their nucleoli have been previously reconstructed in 3-D from serial ultra-thin sections. We used the osmium ammine-B complex (a specific DNA stain) on thick sections of Lowicryl-embedded root fragments. Immunocytochemical techniques using anti-DNA antibodies and rDNA/rDNA in situ hybridization were also applied on ultra-thin sections. We showed on tilted images that the OA-B stains DNA throughout the whole thickness of the section. In addition, very low quantities of cytoplasmic DNA were stained by this complex, which is now the best DNA stain used in electron microscopy. Within the nucleoli the DNA was localized in the fibrillar centers, where large clumps of dense chromatin were also visible. In the two plant species intranucleolar chromatin forms a complex network with strands partially linked to chromosomal nucleolar-organizing regions identified by in situ hybridization. This study describes for the first time the spatial arrangement of the intranucleolar chromatin in nucleoli of higher plants using high-resolution techniques.     

应用H.E染色的鼻咽癌细胞涂片进行DNA含量测定和EB病毒基因原位杂交的研究

分析鼻咽癌常规 H.E染色细胞涂片分别作 Feulgen染色检测 DNA含量和原位杂交检测 EB病毒编码 RNAs(EBERs)的效果 ;将常规 HE染色的 9例正常鼻咽细胞涂片和 38例鼻咽癌细胞涂片用 1%酸性酒精褪色 ,然后作 Feulgen染色 ,应用图象分析仪测定涂片细胞 DNA含量 ,并将 38例鼻咽癌细胞病例的阳性涂片进行 EBERs原位杂交检测 ;结果显示正常鼻咽细胞均为 DNA二倍体核型 ,38例癌阳性涂片中呈 DNA二倍体者 6例 ,DNA非二倍体者 32例 (84.2 % ) ,癌细胞核 EBERs阳性者 35例 (92 .1% ) ;结果表明 HE染色的鼻咽癌细胞学涂片经褪色后作 Feulgen染色和原位杂交检测 ,能较满意进行 DNA和 EBERs分析 ,表明常规 H.E染色和褪色处理均不影响 Feulgen染色和 EB病毒原位杂交的质量效果。本检测方法可靠、可行 ,适用于常规染色细胞学样本的回顾性研究和随访研究 ,并可用于鼻咽癌可疑病人的诊断和鉴别诊断     

An improved method for chromosome-specific labeling of alpha satellite DNA in situ by using denatured double-stranded DNA probes as primers in a primed in situ labeling (PRINS) procedure.

An improved primed in situ labeling (PRINS) procedure that provides fast, highly sensitive, and nonradioactive cytogenetic localization of chromosome-specific tandem repeat sequences is presented. The PRINS technique is based on the sequence-specific annealing in situ of unlabeled DNA. This DNA then serves as primer for chain elongation in situ catalyzed by a DNA polymerase. If biotin-labeled nucleotides are used as substrate for the chain elongation, the hybridization site becomes labeled with biotin. The biotin is subsequently made visible through the binding of FITC-labeled avidin. Tandem repeat sequences may be detected in a few hours with synthetic oligonucleotides as primers, but specific labeling of single chromosomes is not easily obtained. This may be achieved, however, if denatured double-stranded DNA fragments from polymerase-chain-reaction products or cloned probes are used as primers. In the latter case, single chromosome pairs are stained with a speed and ease (1 h reaction and no probe labeling) that are superior to traditional in situ hybridization. Subsequent high-quality Q banding of the chromosomes is also possible. The developments described here extends the range of applications of the PRINS technique, so that it now can operate with any type of probe that is available for traditional in situ hybridization.     

Characterization of DNA damage in yeast apoptosis induced by hydrogen peroxide, acetic acid, and hyperosmotic shock

Saccharomyces cerevisiae has been reported to die, under certain conditions, from programmed cell death with apoptotic markers. One of the most important markers is chromosomal DNA fragmentation as indicated by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining. We found TUNEL staining in S. cerevisiae to be a consequence of both single- and double-strand DNA breaks, whereas in situ ligation specifically stained double-strand DNA breaks. Cells treated with hydrogen peroxide or acetic acid staining positively for TUNEL assay stained negatively for in situ ligation, indicating that DNA damage in both cases mainly consists of single-strand DNA breaks. Pulsed field gel electrophoresis of chromosomal DNA from cells dying from hydrogen peroxide, acetic acid, or hyperosmotic shock revealed DNA breakdown into fragments of several hundred kilobases, consistent with the higher order chromatin degradation preceding DNA laddering in apoptotic mammalian cells. DNA fragmentation was associated with death by treatment with 10 mM hydrogen peroxide but not 150 mM and was absent if cells were fixed with formaldehyde to eliminate enzyme activity before hydrogen peroxide treatment. These observations are consistent with a process that, like mammalian apoptosis, is enzyme dependent, degrades chromosomal DNA, and is activated only at low intensity of death stimuli.     

Denaturation, renaturation, and loss of DNA during in situ hybridization procedures

With the aim of optimizing in situ hybridization methods, alkaline, acid, and thermal denaturation procedures have been studied for their ability to separate the DNA strands of nuclear DNA and for the DNA losses they induce. Isolated methanol/acetic acid-fixed mouse liver nuclei have been used as a biological object. The results, obtained with acridine orange staining and microfluorometry, show that all denaturations studied lead to almost complete strand separation. Quantitative DNA staining and cytometry indicated that with heat and alkaline denaturation about 40% of the DNA is lost. Acid denaturation led to about 20% DNA loss. For the alkaline denaturation, the DNA retention could be improved to a 20% DNA loss by adding 70% ethanol to the denaturation medium. During hybridization, another 20% DNA loss occurs. When denatured nuclei are brought under annealing conditions, a rapid renaturation of a considerable fraction of the remaining DNA occurs. The extent of renaturation was dependent on the type of denaturation used. For the ethanolic alkaline denaturation, it was estimated to be 35%. Quantitative nonautoradiographic in situ hybridization experiments with acetylaminofluorene-modified mouse satellite DNA showed that alkaline denaturation procedures are superior to the heat and acid denaturation. As proven by acridine orange fluorescence measurements, hybridization conditions can be designed that permit DNA.RNA hybridization under in situ DNA.DNA denaturing conditions. These conditions should be very useful, especially for in situ hybridization with single-stranded RNA probes.     

Highly sensitive immunodetection of DNA on sections with exogenous terminal deoxynucleotidyl transferase and non-isotopic nucleotide analogues.

A new method is described for locating DNA on ultra-thin sections. Sections of aldehyde-fixed, plastic-embedded cells were incubated in a medium containing terminal deoxynucleotidyl transferase (TdT) and various non-isotopic nucleotide analogues. The labeled nucleotides bound to the surface of ultra-thin sections were then visualized by an indirect immunogold labeling technique. The resulting labeling pattern was strongly dependent on the divalent cation used in the TdT medium. The method revealed with great precision the specific DNA-containing structures within Ehrlich tumor cells, even where DNA was present in very low amounts. The method is compatible with all usual fixation and embedding procedures and can be combined with cytochemical methods. The in situ TdT method provides a very useful tool for pinpointing the precise location of DNA within biological material at the ultrastructural level.     

DNA extraction procedure: a critical issue for bacterial diversity assessment in marine sediments

In order to evaluate whether different DNA extraction procedures can affect estimates of benthic bacterial diversity, based on 16S rRNA gene terminal restriction fragment length polymorphism (T-RFLP) fingerprinting technique, we compared two in situ lysis procedures (a SDS-based protocol and a commercial kit for DNA recovery) and one cell-extraction protocol on a variety of marine sediments. Despite the two in situ lysis procedures resulted in significantly different DNA yields (highest with the SDS in situ lysis), estimates of bacterial diversity provided a not significantly different ribotype richness, as well as similar values of the Shannon-Wiener (H') and Margalef (d) indices of biodiversity and of evenness (Pielou index, J). Conversely, the cell-extraction procedure for DNA extraction resulted always in a significantly lower ribotype richness and diversity. The analysis of similarities (anosim) among the T-RFLP electropherograms allowed concluding that ribotypes composition did not change significantly using different protocols. However, the analysis of beta-diversity (turnover diversity) revealed that a large number of ribotypes was observed exclusively with one of the three protocols utilized. When unshared ribotypes from in situ lysis and cell extraction were pooled together, total ribotype richness resulted much higher (up to 80%). Our results indicate that estimates of ribotype diversity based on a single protocol of DNA extraction can significantly underestimate the total number of bacterial ribotypes present in the benthic domain. We recommend that future studies will not only integrate different DNA extraction procedures, but also will explore the possibility of integrating two or more different genetic markers in order to increase our ability to detect the actual bacterial diversity in environmental samples.     

Chromosome structure and DNA replication in nurse and follicle cells of Drosophila melanogaster

In the nurse cells of Drosophila, nuclear DNA is replicated many times without nuclear division. Nurse cells differ from salivary gland cells, another type of endoreplicated Drosophila cell, in that banded polytene chromosomes are not seen in large nurse cells. Cytophotometry of Feulgen stained nurse cell nuclei that have also been labeled with ³H-thymidine shows that the DNA contents between S-phases are not doublings of the diploid value. In situ hybridization of cloned probes for 28S+18S ribosomal RNA, 5S RNA, and histone genes, and for satellite, copia, and telomere sequences shows that satellite and histone sequences replicate only partially during nurse cell growth, while 5S sequences fully replicate. However, during the last nurse cell endoreplication cycle, all sequences including the previously under-replicated satellite sequences replicate fully. In situ hybridization experiments also demonstrate that the loci for the multiple copies of histone and 5S RNA genes are clustered into a small number of sites. In contrast, 28S+18S rRNA genes are dispersed. We discuss the implications of the observed distribution of sequences within nurse cell nuclei for interphase nuclear organization. — In the ovarian follicle cells, which undergo only two or three endoreplication cycles, satellite, histone and ribosomal DNA sequences are also found by in situ hybridization to be underrepresented; satellite sequences may not replicate beyond their level in 2C cells. Hence the pathways of endoreplication in three cell types, salivary gland, nurse, and follicle cells, share basic features of DNA replication, and differ primarily in the extent of association of the duplicated chromatids.     

Staining of depolymerised DNA in mammalian tissues with methyl violet 6B and crystal violet

The paper contains an account of DNA staining with basic dyes; methyl violet 6B and crystal violet in mammalian tissue sections after RNA extraction with cold concentrated phosphoric acid. The study shows that the best staining is obtained at pHs 2.5 and 3.5. Dehydration of stained nuclei is perfect when a mixture of absolute ethanol and n-butanol is used followed by treatment of sections in isoamyl or amyl alcohol. The in situ absorption data of nuclei stained with aqueous solution of methyl violet 6B as well as with crystal violet are also presented. Possible mechanism of staining as well as an explanation for dye-leaching when sections are dehydrated through ethanol are discussed.     

Detection of human papillomavirus in cervical smears. A comparison of in situ hybridization, immunocytochemistry and cytopathology

A comparison of different methods for the detection of cervical human papillomavirus (HPV) infection was made on patients attending the cervical dysplasia clinic. Cytomorphology, immunocytochemistry and in situ hybridization were compared for their ability to detect HPV. Separate cervicovaginal smears from 50 patients were tested for HPV types 6/11, 16 and 18 by in situ hybridization using 35S-labeled DNA probes. Duplicate smears from the same patients were Papanicolaou stained and evaluated for evidence of condylomatous and dysplastic changes. Twenty-five matching cervical biopsies were immunostained for HPV capsid antigen and tested by in situ hybridization for HPV DNA. The cytologic smears of 20 patients (40%) were positive for HPV DNA. Six patients had HPV 6/11, ten had HPV 16, three had HPV 18, and one had both HPV 6/11 and HPV 16. There was a high correlation between condylomatous cytopathology and antigen and DNA detection. One-third of the specimens with condylomatous changes were DNA negative by the tested probes, suggesting the presence of other HPV types in the genital tract.     

Microwave irradiation-stimulated in situ hybridization procedure with biotinylated DNA probe.

A microwave-stimulated in situ hybridization technique using biotin-labeled DNA probe is described. Both hybridization reaction and the detection of the biotin label (with a alkaline phosphatase or immunofluorescence method) has been performed in the microwave oven. All procedures are completed within one hour. The described method was applied for identification of nucleic acid sequences of human immunodeficiency virus in human cell lines. The resolution and the intensity of the signal are as good as from a standard technique with overnight incubation of the probe. Because of the simplicity and speed of the technique, this procedure can be used in a number of other applications.     

Losses of material during cytological preparation of nuclei and chromosomes

Losses of nucleic acids and protein from cells and nuclei during the procedures used for in situ hybridization and other ‘denaturation-renaturation techniques’ were determined both by chemical analysis and, in case of DNA, also by measurements of radioactivity after labelling with 3H-thymidine (scintillation counting and autoradiography). The electron microscopic appearance of such preparations was also examined. Losses of DNA are considerable, with some variation according to the specific treatment applied, and suggest that results obtained with such procedures are not interpretable in quantitative terms.     

Thermal denaturation of DNA in situ as studied by acridine orange staining and automated cytofluorometry

Thermal denaturation of nuclear DNA is studied in situ in individual cells or isolated cell nuclei by employing the property of the fluorochrome acridine orange (AO) to differentially stain native and denatured DNA and by using an automated flow-through cytofluorimeter for measurement of cell fluorescence. RNAse-treated cells, or cell nuclei, are heated, stained and measured while in suspension and AO-DNA interaction is studied under equilibrium conditions. Measurements are made rapidly (200 cells/sec); subpopulations of cells from a measured sample can be chosen on the basis of differences in their staining or light-scattering properties and analysed separately. DNA denaturation in situ is rapid; it approaches maximum during the first 5 min of cell heating. Divalent cations stabilize DNA against denaturation. At low pH the transition occurs at lower temperature and the width of the transition curves (‘melting profiles’) is increased. Decrease in ionic strength lowers the DNA melting temperature. This effect is much more pronounced in cells pretreated with acids under conditions known to remove histones. Histones thus appear to stabilize DNA in situ by providing counterions. At least four separate phases can be distinguished in melting profiles of DNA in situ; they are believed to indicate different melting points of DNA in complexes with particular histones. A decrease in cell (nuclear) ability to scatter light coincides with DNA melting in situ, possibly representing altered refractive and/or reflective properties of cell nuclei. Formaldehyde, commonly used to prevent DNA renaturation, is not used in the present method. The heat-induced alterations in nuclear chromatin are adequately stabilized after cell cooling in the absence of this agent. Cells heated at 60–85 °C exhibit increased total fluorescence after AO-staining, which is believed to be due to unmasking of new sites on DNA. This increase is neither correlated with DNA melting, nor with the presence of histones. Possibly, it reflects destruction of DNA superstructure maintained at lower temperatures by DNA associations with other than histone macromolecules (nuclear membrane).