Quantitative aspects of the cytochemical Feulgen-DNA procedure studied on model systems and cell nuclei

Quantitative aspects of DNA losses during fixation and pararosaniline(SO2)-Feulgen staining of microscopic preparations were studied. The preparation of a new cytochemical model, consisting of DNA-protein layers (with thicknesses between 0.1 and 5.0 micrometer) on microscopic glass slides is described and potentialities and limitations of this model are discussed. Polyacrylamide films into which high molecular weight calf thymus DNA or chicken erythrocyte nuclei had been constrained served as another model. As biological objects chicken erythrocyte nuclei and rat liver nuclei either in suspension or on microscopical glass slides were used. The experimental results indicate a loss of about 5% of the DNA due to the fixation procedure applied. Hydrolysis in 5 N HCl at room temperature, staining with the pararosaniline-Schiff medium and rinsing with sulfurous acid induced losses of DNA too, varying in amount depending on the type of preparation used. About 10% of the original DNA content is lost in total from chicken erythrocyte nuclei and rat liver nuclei dried on microscopical glass slides, from chicken erythrocyte nuclei constrained in polyacrylamide films, and from DNA-protein layers on microscopic glass slides. For nuclei fixed and stained in suspension the total losses amount to about 40%. The differences in losses between various types of preparations are discussed. Biochemically, the content of DNA originally present per chicken erythrocyte nucleus was determined to be 2.52 pg, a value, which is in good accordance with reliable biochemical data published already. It is shown that calibration of cytochemical staining intensities into biochemical units or absolute amounts of material by use of a model system, is only reliable when it is known or to be expected that both the loss of material due to fixation and staining, and the stoichiometric relation between material present and dye molecules is identical. The same holds for the application of internal biological reference systems.     

Spreading and staining of human metaphase chromosomes on aminoalkylsilane-treated glass slides

Summary The properties of aminoalkylsilane-treated glass slides for the preparation of metaphase spreads and their staining quality have been studied and compared with those of slides which had only been cleaned in ethanol/ether. The parameters investigated were: (1) the average area of metaphases from cultures of blood from both healthy donors and haematology patients; (2) the influence of the positively charged coating on the quality of quinacrine- and Giemsa-banding patterns; (3) non-specific background staining for these banding methods; (4) the number of metaphases as compared to the number of interphase cell nuclei per area of preparation; and (5) the Feulgen-staining intensities of chromosomes and chicken erythrocyte nuclei.The quality of metaphase preparations and the differential staining of chromosomes is better on aminoalkysilane-treated glass slides than that of preparations on routinely cleaned normal microscope slides. In the preparations on aminoalkylsilance-treated slides, the distribution of the cells over the glass surface is more homogeneous; and no influence could be detected on the relative frequency of metaphases as compared to the number of non-divided cell nuclei; the average area per metaphase is increased by about 10% and consequently the number of overlapping chromosomes is decreased.Preparations on aminoalkylsilane-treated glass, after Q-, G- and DAPI-banding procedures, always showed less binding of the staining compounds to the glass slide (a cleaner background) than those on routinely cleaned microscope glass slides. The Feulgen-pararosaniline staining intensities of human metaphase chromosomes and chicken erythrocyte nuclei are the same on aminoalkylsilane-treated slides and on routinely cleaned glass slides. Furthermore, the reproducibility and constancy of quinacrine banding was improved by development of an equilibrium staining method which does not require a washing procedure. The medium, containing 0.002% quinacrine, allows optimal staining results to be obtained for microphotography purposes within 30 min of staining (for visual inspection at least 90 min is required) and is used as the embedding medium.In combination with aminoalkylsilane-treated glass slides, this procedure leads to a clean background and reproducible banding patterns of excellent quality, the results being better and more constant than those of methods described before.     

Quantitative cytochemical determination of desoxyribonucleic acid with the Feulgen nucleal reaction

The possibility of using the Feulgen nucleal reaction for a quantitative cytochemical estimation of desoxyribonucleic acid (DNA) was investigated. The intensity of the reaction in nuclei was determined by absorption measurements with the microscope. The accuracy of such measurements was tested by comparison with measurements on the same material with a Beckman spectrophotometer. The values obtained with the microscope agreed within a few per cent with those obtained with the Beckman spectrophotometer. Furthermore, the errors introduced by uneven distribution of absorbing material, by variations in the numerical aperture of the system, and by variation in the area used on the phototube were investigated empirically. The following variables were studied with regard to their effect on the intensity of the Feulgen reaction: type of fixation, time of hydrolysis after acetic acid-alcohol and formalin fixation, time of staining in leucobasic fuchsin, method of preparation of leucobasic fuchsin. The intensity of the Feulgen reaction in liver and erythrocyte nuclei of various vertebrates, fixed in acetic acid-alcohol, was then compared with the DNA content of these nuclei as determined by chemical analysis on a known number of nuclei. The intensity of the reaction was found to be proportional to the DNA content of the nuclei, if nuclei of similar structure and DNA concentration were compared. In nuclei of different structure and DNA concentration (i.e. liver and erythrocyte nuclei), fixed in acetic acid-alcohol, the intensity of the Feulgen reaction was, however, not proportional to the DNA content. This difficulty was overcome by isolating nuclei in sucrose and by fixing them in formalin. Uniform distribution of DNA and therefore uniform coloring after the Feulgen reaction were thus obtained. In such nuclei with uniform distribution of absorbing material the Feulgen reaction was found to be proportional to the DNA content of nuclei, even if they differed greatly in their DNA concentration. The Feulgen nucleal reaction is not quantitative in an absolute sense. For absolute determinations nuclei of known DNA content must be treated together with the unknown material to serve as standard. From these data it therefore appears possible to determine cytochemically relative amounts of DNA in cellular structures by measuring their absorption after treatment with the Feulgen nucleal reaction.     

How sensitive may cytochemical methods detect alterations of the glycocalyx?

This review reports on the relationship of light and electron microscopic cytochemical staining and chemo-affinity labelling to the conformational state of the human erythrocyte glycocalyx glycophorin. According to a recent suggestion, primary glycophorin conformation is associated with binding to the lipid bilayer of this glycoprotein's N-terminal segment. Pretreatment of cells prior to fixation may cause this segment of detach from the lipid lamella and to acquire a less orientation. Alterations of the glycocalyx were sensitively demonstrated by topo-optical staining and by the labelling of unmasked binding sites.     

Disruption of the typical chromatin structure in a 2500 base-pair region at the 5' end of the actively transcribed ovalbumin gene.

We examined the chromatin organizations of approximately 3 kb of DNA in the 5'-end flanking region of the ovalbumin gene in chicken erythrocyte and oviduct cell nuclei. With specific DNA probes and an indirect end-labeling technique, we analysed the pattern of the DNA fragments obtained after micrococcal nuclease digestion and generated comparative maps of the nuclease cuts. This region of the chicken genome displays a "typical" chromatin arrangement in erythrocyte nuclei, with nucleosomes apparently positioned at random. In contrast, in oviduct nuclei, the same region has an "altered" chromatin structure, and lacks a typical nucleosomal array. The existence of specifically positioned proteins and of alterations in the DNA secondary structure in this region of the oviduct chromatin is suggested by comparison of the nuclease cleavage maps which reveals specific changes: disappearance of nuclease cuts present in "naked" and erythrocyte chromatin DNAs, and appearance of new cuts absent from these DNAs.     

Structural repeat units of Chinese hamster ovary chromatin. Evidence for variations in repeat unit DNA size in higher eukaryotes.

DNA lengths in the structural repeat units of Chinese hamster ovary (CHO) and chicken erythrocyte chromatin were compared by analyzing the sizes of DNA fragments produced after treatment of nuclei with staphylococcal nuclease. The repeat length of CHO chromatin (173 +- 4 BP) is about 20 base pairs (BP) smaller than that of chicken erythrocyte chromatin (194 +- 8 BP). Repeat lengths of rat liver and calf thymus chromatin were found to be about 10 BP shorter than that of chicken erythrocyte chromatin. Thus significant variations occur in repeat units of chromatin of higher eukaryotes. These variations occur in the lengths of "spacer" (or "internucleosomal") DNA segments, not in "core particle" (or "nucleosomal") DNA lengths. The concept of spacer regions and the possible influence of H1 histones is discussed.     

Cytochemical hybridization with fluorochrome-labeled RNA. II. Applications

The cytochemical detection of specific DNA sequences by hybridization with fluorochrome-labeled RNA and detection of the hybrids by fluorescence microscopy is described. RNAs complementary to the DNA of the kinetoplasts of Crithidia luciliae (an insect trypanosome) or to adenovirus-5 (Ad-5) DNA were labeled with the hydrazine derivative of tetramethylrhodamine isothiocyanate (TRITC). The specificity of the reactions between the complementary RNAs labeled both with 3H and tetramethylrhodamine was studied by cross-hybridization experiments using a model system in which the DNAs were bound to Sepharose beads. The extent of the reaction was measured by scintillation counting of the bead suspensions and quantitative fluorescence microscopy of individual Sepharose beads. The ability of the rhodamine-labeled cRNAs to hybridize and the absence of interference of the fluorochrome label with the specificity of the hybridization reaction was thus demonstrated. After cytochemical hybridization on microscopic preparations of C. luciliae cells the rhodamine-labeled kinetoplast cRNA stains only the kinetoplasts. No fluorescence was observed in the nuclei. After cytochemical hybridization of rhodamine-labeled Ad-5 cRNA with virus infected KB cells a distinct staining pattern in the nuclei was observed. No fluorescence was seen in uninfected cells, or after hybridization with heterologous rhodamine-labeled RNA. The possibilities and limitations of cytochemical hybridization with rhodamine-labeled RNA are discussed.     

The use of methyl green-pyronin staining after glutaraldehyde fixation and paraffin or araldite embedding.

Methyl green-pyronin staining has been used for localization of RNA and DNA in chick and mouse embryonic tissues and in insect larval salivary glands. Glutaraldehyde or tricholoracetic acid-lanthanum acetate (TCA-LA) was used as fixative and paraffin wax or Araldite was used as embedding medium. For good results the following are specially desirable: fixation with 2.5% glutaraldehyde, dehydration in alcohols for short time, and the use of fresh staining solutions. After TCA-LA fixation the final results are much less specific. The digestion with RNAse appears essential for the detection of RNA because pyronin does not seem to be entirely specific to RNA. The results show that glutaraldehyde a common fixative for electron microscopic work, is particularly suitable fixative for light microscopic cytochemical investigations if followed by methyl green-pyronin staining; furthermore, methyl green-pyronin staining after glutaraldehyde fixation can be carried out on Araldite sections.     

Comparison of the Papanicolaou and Feulgen staining methods for DNA quantification by image analysis.

The possibility of using archival cytology material to study the evolution of neoplastic disease with regard to DNA content abnormalities was investigated. The accuracy of measuring the integrity optical density (OD) of nuclei that correlates to DNA amounts of those nuclei, on slides stained by the Papanicolaou method, was assessed and compared with a standard Feulgen method. Our data on rat liver nuclei peritoneal washings from patients with ovarian cystadenofibromas and ovarian cystadenocarcinomas suggested that analysis of cytological material using the Papanicolaou method is not reliable and that destaining the slides followed by Feulgen staining provides an optimal and reliable method of DNA quantification.     

Light microscopical detection of antigens and lectin binding sites with gold-labelled reagents on semi-thin Lowicryl K4M sections: Usefulness of the photochemical silver reaction for signal amplification

Summary In the present study, we have investigated the applicability of semi-thin sections from low temperature Lowicryl K4M-embedded tissues for cytochemical labelling with protein A—gold and lectin—gold complexes. In order to ensure the best possible signal-to-noise ratio antibodies, protein A—gold and lectin—gold were applied in concentrations used for labelling at the electron microscope level. Furthermore, due to the lack of an appropriate chemical procedure for resin removal, untreated semi-thin sections were incubated. Under such conditions, semi-thin sections displayed either no visible staining or only a faint incomplete staining. However, following photochemical silver reaction, the latent or faint incomplete staining was rendered visible in most cases. It is concluded that the same block of Lowicryl K4M-embedded tissue and the same labelling reagents can be used for both light and electron microscopical cytochemical studies. At the light microscopical level, a high degree of structural and specific staining information is obtained. The reactivity of cellular components with antibodies or lectins is preserved even after years of storage of the blocks or slides containing semi-thin sections.     

Glass slide models for immunocytochemistry and in situ hybridization

We have developed a model system based on the immobilization of microdroplets (0.1 l volumes) of antigens or of target (sense) oligonucleotides onto aminoalkylsilane-coated glass slides. Oligopeptide antigens need to be vapour-fixed in order to achieve efficient immobilization, while oligonucleotides do not require fixation. Protein antigens, exemplified by rabbit immunoglobulin, may be subjected to liquid fixation. The glass slide models are optically translucent and useful both for in situ hybridization and immunocytochemistry. The slides are compatible with detection systems of both light and fluorescence microscopy and permit measurement of staining intensities by microfluorimetry or computerized microdensitometry. The model systems can be used for comparisons of method sensitivities, for characterizing antibody and probe sensitivities and cross-reactivities, and as internal standards or quality controls for immunocytochemistry and in situ hybridization.     

Interaction of poly-L-lysine with chromatin. Inhibition of in situ priming for Escherichia coli DNA polymerase

The priming capacity of chromatin of fixed nuclei and chromosomes for exogenous DNA polymerase can be evaluated radioautographically by the incorporation of labeled nucleotides. It had previously been reported that acid fixation or acid treatment of alcohol-fixed tissues led to increased priming when calf thymus DNA polymerases, specific for single-stranded DNA, were used. We employed Escherichia coli DNA polymerase and sequential treatments of the fixed tissue with acid and poly-L-lysine in order to elucidate the mechanism through which the acid effect is produced. Acid treatment enhanced chromatin priming for the E coli DNA polymerase, and saturation of the chromatin with poly-L-lysine strongly inhibited the reaction. This inhibition was reversible through subsequent treatment with acid. Wide differences in priming were observed between cell types of alcohol-fixed chicken blood smears: thrombocyte and lymphocyte nuclei exhibited strong priming ability whereas erythrocyte nuclei failed to support any detectable priming. We conclude that the acid effect is readily interpretable in terms of acid-mediated changes in the association between DNA and protein in the chromatin complex.     

The presence of the 30 nm filament structure of chromatins in intact chicken erythrocytes observed by 31P NMR

A 31P NMR spectrum of chicken erythrocyte chromatins in vivo was obtained. The spectrum was characterized by examining the spectra of isolated nuclei and nondigested chromatins under various conditions. The results have shown that most chromatins in chicken erythrocyte nuclei assume the 30 nm filament structure as a second step of condensation of DNA in vivo.     

A comparison of internal standards for plant cytophotometry

Plant cell nuclei were compared with chicken erythrocyte nuclei for use as internal standards for microspectrophotometry. The amount of DNA per nucleus and the coefficient of variation for measurement of individual nuclei were determined for cells from dormant embryos of Pinus taeda and Pinus coulteri, from onion root tips and from chicken erythrocytes. The chicken erythrocytes had the least variability and thus were best suited for use as a standard. Onion root tips were least suitable, with a coefficient of variation 2 1/2 times that of erythrocytes. Although onion root tips have been used as an internal standard in other studies, their mitotic activity, in contrast with the nonreplication of DNA of mature erythrocytes, is reflected in a broad distribution of nuclei with values in the 2C-4C range. Coulter pine mature embryos were at the 3C level, whether dry or hydrated, while loblolly pine embryos were in the 2C state. This confirms previous reports. The coefficient of variability for the pine embryo cells was 1 1/2 times that of erythrocytes for nonhydrated seeds and twice the erythrocyte value for hydrated seeds. The larger 2C values for pine (26 pg for P. taeda and 17 pg for P. coulteri) are closer to values expected for many plant species than the 3 pg level of the chicken erythrocytes. Dormant P. taeda embryo cells (2C) are suggested as an alternative where the experimental material has large DNA values and/or chicken erythrocytes are difficult to procure. Large sample size is recommended for the plant materials if they are to be used as internal standards in Feulgen cytophotometry.     

Cytopress: automated slide preparation of cytologic material from suspension

This paper describes a new automated system to prepare slides of cytological material from suspension. The system collects material on a filter tape by filtration and transfers it to glass slides by means of pressure-fixation. Using cervical cells as a model, results show that a well-defined cell number is evenly deposited over a standardized area, while a small number of cells is retained on the tape and a negligible number lost in the filtrate. Contamination is very small. Application of the system to other cytological material (fine needle aspirations, monolayer and cell suspension cultures, agar cultures, and isolated nuclei) is shown. In general, more than one slide can be made from one sample. Several histological staining procedures as well as immunofluorescence labeling protocols can be applied to the preparations obtained in this way. This system thus introduces a method that will standardize specimen preparation, is quick, saves operator time, and can be used for both diagnostic and research applications.     

Linker histone subtype composition and affinity for chromatin in situ in nucleated mature erythrocytes

The replacement linker histones H1(0) and H5 are present in frog and chicken erythrocytes, respectively, and their accumulation coincides with cessation of proliferation and compaction of chromatin. These cells have been analyzed for the affinity of linker histones for chromatin with cytochemical and biochemical methods. Our results show a stronger association between linker histones and chromatin in chicken erythrocyte nuclei than in frog erythrocyte nuclei. Analyses of linker histones from chicken erythrocytes using capillary electrophoresis showed H5 to be the subtype strongest associated with chromatin. The corresponding analyses of frog erythrocyte linker histones using reverse-phase high performance liquid chromatography showed that H1(0) dissociated from chromatin at somewhat higher ionic strength than the three additional subtypes present in frog blood but at lower ionic strength than chicken H5. Which of the two H1(0) variants in frog is expressed in erythrocytes has thus far been unknown. Amino acid sequencing showed that H1(0)-2 is the only H1(0) subtype present in frog erythrocytes and that it is 100% acetylated at its N termini. In conclusion, our results show differences between frog and chicken linker histone affinity for chromatin probably caused by the specific subtype composition present in each cell type. Our data also indicate a lack of correlation between linker histone affinity and chromatin condensation.     

DNA-protein binding in interphase chromosomes

The metachromatic dye, azure B, was analyzed by microspectrophotometry when bound to DNA fibers and DNA in nuclei with condensed and dispersed chromatin. The interaction of DNA and protein was inferred from the amount of metachromasy (increased β/α-peak) of azure B that resulted after specific removal of various protein fractions. Dye bound to DNA-histone fibers and frog liver nuclei fixed by freeze-methanol substitution shows orthochromatic, blue-green staining under specific staining conditions, while metachromasy (blue or purple color) results from staining DNA fibers without histone or tissue nuclei after protein removal. The dispersed chromatin of hepatocytes was compared to the condensed chromatin of erythrocytes to see whether there were differences in DNA-protein binding in "active" and "inactive" nuclei. Extraction of histones with 0.02 N HCl, acidified alcohol, perchloric acid, and trypsin digestion all resulted in increased dye binding. The amount of metachromasy varied, however; removal of "lysine-rich" histone (extractable with 0.02 N HCl) caused a blue color, and a purplish-red color (µ-peak absorption) resulted from prolonged trypsin digestion. In all cases, the condensed and the dispersed chromatin behaved in the same way, indicating the similarity of protein bound to DNA in condensed and dispersed chromatin. The results appear to indicate that "lysine-rich" histone is bound to adjacent anionic sites of a DNA molecule and that nonhistone protein is located between adjacent DNA molecules in both condensed and dispersed chromatin.     

Positive and negative regulation of replication in hybrid cells]

DNA replication blockage in various differentiated cells was investigated on the model of heterokaryons. Two distinct types of DNA synthesis regulation in heterokaryons "differentiated cell + proliferating cell" were revealed: I. Neutrophils and nucleated erythrocytes efficiently prevented the entry of non-malignant proliferating cells nuclei into the S-period but usually failed to substantially inhibit the replication in malignant cells nuclei. Both "mortal" and immortalized proliferating cells activated the DNA synthesis in neutrophil and chicken erythrocyte nuclei. II. Macrophages did not influence the DNA synthesis in the nuclei of non-malignant cells in heterokaryons but drastically inhibited that in the nuclei of malignant cells. Only immortalized cells reactivated DNA synthesis in the nuclei of macrophages. These data show that the mechanisms maintaining differentiated cells in non-proliferating state are not uniform. Nucleated erythrocytes were shown to suppress the duplication of centrioles in partner cells. The possibility of the blockage of DNA replication upon the fusion of two proliferating cells (fibroblast + leukemia cell) was demonstrated for the first time in the present work. The influence of various oncogenes upon the regulation of DNA synthesis in heterokaryons was investigated in detail. New modifications of the methods of cell fusion, enucleation and heterokaryon identification were proposed.