Development of a binding assay for p53/HDM2 by using homogeneous time-resolved fluorescence

Using in situ hybridization techniques, a fixation step must precede denaturation to prevent disintegration of the chromosomes. The analysis of nuclei fixed by paraformaldehyde, preserving the native structure (three-dimensional or 3D fixation and analysis) has become possible with the development of confocal microscopy; however, the analysis of those fixed by methanol and acetic acid, dehydrating the nuclei (two-dimensional or 2D fixation and analysis), remains a very valuable tool for practical use in diagnostics and also in many cases for research. We compared both types of fixation and analyses using different cell lines and different DNA probes. Fixation of cells by methanol and acetic acid leads to the enlargement of contact of nuclei with the slide surface, resulting in a substantial increase of nuclear diameter, flattening of the nucleus, and consequently to a distortion of gene topology. Our results indicate that chromatin structures located in the outer parts of the nuclear volume (e.g., heterochromatin of some centromeres) are relatively shifted to the membrane of these nuclei, keeping the absolute centromere-membrane distance constant. On the other hand, euchromatin located in the inner parts of the nuclear volume is not shifted outside proportionally to the increase of molecular dimensions; consequently, the relative distances for the center of nucleus to gene are smaller after methanol-acetic acid fixation. The limitations of the analysis of dehydrated preparations for practical use and in research are discussed.     

Evidence from studies on segregated nucleoli that nucleolar silver staining proteins C23 and B23 are in the fibrillar component

Several procedures for the silver staining of nucleoli have been evaluated at the electron microscopic level to determine optimal conditions for ultrastructural preservation and staining specificity. The present study shows that a brief fixation with 1% buffered formaldehyde followed by methanol: acetic acid (3 : 1) fixation yielded optimal preservation and silver staining of nucleoli. Using this procedure for electron microscopic studies of interphase nucleoli, it was found that the punctate silver grains observed by light microscopy were composed of fine silver granules, of approx. 100 Å diameter, organized in discrete clusters. In similar studies on adriamycin-induced segregated nucleoli, it was observed that the silver staining reaction was mainly limited to the fibrillar portion of the nucleolus. Accordingly, nucleolar proteins C23 and B23, found earlier to be the major silver binding proteins of the nucleolus, are mainly concentrated in the fibrillar nucleolar component.     

The effect of different fixatives on chromatin: Cytochemical and ultrastructural approaches

Summary This study explores the effects of two types of fixative on chromatin. The first type (acrolein, glutaraldehyde) engenders a high degree of ultrastructural preservation. The other type are fixatives that are widely used in cytochemistry and cytogenetics (acetic acid, 31 by vol. methanol-acetic acid, methanol alone, formaldehyde).Lymphocytes of adult rats so-fixedin vitro were prepared for electron microscopy or microdensitometric evaluations of smears. Assessments were made of variations in their total protein, nuclear basic protein and DNA contents. DNA was determined both as Feulgen-positive material and by its binding of intercalating dyes (Methyl Green, specific for double-stranded polynucleotides).Our results showed that some fixatives break up the chromatin organization by acting on particular components of chromatin fibres. They can thus be considered to be destructive agentsin situ. In addition, a revaluation of some aldehyde fixatives is proposed for both ultrastructural and cytochemical research.     

Processing deep-sea particle-rich water samples for fluorescence in situ hybridization: consideration of storage effects, preservation, and sonication

Particles are often regarded as microniches of enhanced microbial production and activities in the pelagic ocean and are vehicles of vertical material transport from the euphotic zone to the deep sea. Fluorescence in situ hybridization (FISH) can be a useful tool to study the microbial community structures associated with these particles, and thus their ecological significance, yet an appropriate protocol for processing deep-sea particle-rich water samples is lacking. Some sample processing considerations are discussed in the present study, and different combinations of existing procedures for preservation, size fractionation sequential filtration, and sonication were tested in conjunction with FISH. Results from this study show that water samples should be filtered and processed within no more than 10 to 12 h after collection, or else preservation is necessary. The commonly used prefiltration formaldehyde fixation was shown to be inadequate for the rRNA targeted by FISH. However, prefiltration formaldehyde fixation followed by immediate freezing and postfiltration paraformaldehyde fixation yielded highly consistent cell abundance estimates even after 96 days or potentially longer storage. Size fractionation sequential filtration and sonication together enhanced cell abundance estimates by severalfold. Size fractionation sequential filtration effectively separated particle-associated microbial communities from their free-living counterparts, while sonication detached cells from particles or aggregates for more-accurate cell counting using epifluorescence microscopy. Optimization in sonication time is recommended for different specific types of samples. These tested and optimized procedures can be incorporated into a FISH protocol for sampling in deep-sea particle-rich waters.     

Processing Deep-Sea Particle-Rich Water Samples for Fluorescence In Situ Hybridization: Consideration of Storage Effects, Preservation, and Sonication

Particles are often regarded as microniches of enhanced microbial production and activities in the pelagic ocean and are vehicles of vertical material transport from the euphotic zone to the deep sea. Fluorescence in situ hybridization (FISH) can be a useful tool to study the microbial community structures associated with these particles, and thus their ecological significance, yet an appropriate protocol for processing deep-sea particle-rich water samples is lacking. Some sample processing considerations are discussed in the present study, and different combinations of existing procedures for preservation, size fractionation sequential filtration, and sonication were tested in conjunction with FISH. Results from this study show that water samples should be filtered and processed within no more than 10 to 12 h after collection, or else preservation is necessary. The commonly used prefiltration formaldehyde fixation was shown to be inadequate for the rRNA targeted by FISH. However, prefiltration formaldehyde fixation followed by immediate freezing and postfiltration paraformaldehyde fixation yielded highly consistent cell abundance estimates even after 96 days or potentially longer storage. Size fractionation sequential filtration and sonication together enhanced cell abundance estimates by severalfold. Size fractionation sequential filtration effectively separated particle-associated microbial communities from their free-living counterparts, while sonication detached cells from particles or aggregates for more-accurate cell counting using epifluorescence microscopy. Optimization in sonication time is recommended for different specific types of samples. These tested and optimized procedures can be incorporated into a FISH protocol for sampling in deep-sea particle-rich waters.     

Alcoholic Bouin fixation of insect nervous systems for Bodian silver staining. II. Modified solutions

A previously devised synthetic equivalent of 'aged alcoholic Bouin (Duboscq-Brasil) fixative was modified in various ways to discover which of the chemical changes brought about by aging were important in improving fixation and staining. Effects were tested with ventral nerve cord ganglia of the cockroach Periplaneta americana, locust Schistocerca gregaria, and honey bee Apis mellifera. Formation of reaction products, chiefly ethyl acetate and diethoxymethane, seemed to play only a subsidiary role: neither individually appeared essential as long as a sufficient quantity of one or the other was present. In place of diethoxymethane, ethyl acetate concentration could be increased to 25% with little effect on results. Reduction in concentration of two of the original constituents, formaldehyde and ethanol, appeared to be the principal factor in improving fixation. Varying the concentration of each original constituent individually revealed that formaldehyde mainly increased glial staining, ethanol increased tissue shrinkage and reduced overall staining intensity, acetic acid improved preservation, and picric acid decreased glial staining but produced few other effects within a wide range of concentrations, though its omission seriously impaired overall preservation and staining. Varying the ethanol and acetic acid concentrations simultaneously confirmed that they acted in opposite ways. A decrease in ethanol and an increase in acetic acid both improved results. The optimum mixture, 'improved synthetic alcoholic Bouin' (40% formaldehyde 0-15: ethanol 25: acetic acid 5: ethyl acetate 5: diethoxymethane 15: picric acid 0.5: water to 100), gives better preservation and more intense staining, and formaldehyde content can be varied to give the degree of glial staining and more intense staining, and formaldehyde content can be varied to give the degree of glial staining required. Without formaldehyde glial staining is virtually eliminated, while preservation and staining of the neurons appears unaffected. This modification seems to offer a valuable advance in technique.     

Alcoholic Bouin Fixation of Insect Nervous Systems for Bodian Silver Staining. II. Modified Solutions

A previously devised synthetic equivalent of 'aged' alcoholic Bouin (Duboscq-Brasil) fixative was modified in various ways to discover which of the chemical changes brought about by aging were important in improving fixation and staining. Effects were tested with ventral nerve cord ganglia of the cockroach Periplaneta americana, locust Schistocerca gregaria, and honey bee Apis mellifera. Formation of reaction products, chiefly ethyl acetate and diethoxymethane, seemed to play only a subsidiary role: neither individually appeared essential as long as a sufficient quantity of one or the other was present. In place of diethoxymethane, ethyl acetate concentration could be increased to 25% with little effect on results. Reduction in concentration of two of the original constituents, formaldehyde and ethanol, appeared to be the principal factor in improving fixation. Varying the concentration of each original constituent individually revealed that formaldehyde mainly increased glial staining, ethanol increased tissue shrinkage and reduced overall staining intensity, acetic acid improved preservation, and picric acid decreased glial staining but produced few other effects within a wide range of concentrations, though its omission seriously impaired overall preservation and staining. Varying the ethanol and acetic acid concentrations simultaneously confirmed that they acted in opposite ways. A decrease in ethanol and an increase in acetic acid both improved results. The optimum mixture, 'improved synthetic alcoholic Bouin' (40% formaldehyde 0-15: ethanol 25: acetic acid 5: ethyl acetate 5: diethoxymethane 15: picric acid 0.5: water to 100), gives better preservation and more intense staining, and formaldehyde content can be varied to give the degree of glial staining required. Without formaldehyde glial staining is virtually eliminated, while preservation and staining of the neurons appears unaffected. This modification seems to offer a valuable advance in technique.     

Choice of fixation and denaturation for the triple labelling of intra-cytoplasmic antigen,bromodeoxyuridine and DNA

A triple staining method of intra-cytoplasmic antigen, bromodeoxyuridine (BrdU), and DNA for fluorescence image analysis is described. Several kinds of fixation and DNA denaturation methods were tested to obtain a technique suitable for heterogeneous tissues. The model chosen was the analysis of plasma cells in bone marrow. The fluorochromes used were fluorescein isothiocyanate (FITC) for intra-cytoplasmic antigens (light chain immunoglobulins), aminomethylcoumarin acetic acid (AMCA) for BrdU, and propidium iodide (PI) for DNA. The quality of the staining was analysed according to: (1) cell morphology with a good preservation of the chromatin structure, (2) intensity of light chains and of BrdU labelling, and (3) the quality of DNA staining judged from a DNA histogram. For most of the analysed tissues, fixation with methanol followed by 0.5% paraformaldehyde and denaturation by an NaOH concentration adapted to the tissue gave good results. However, in our model fixation by methanol, followed by methanol/acetic acid and denaturation of DNA by 0.03 N NaOH was the solely satisfactory technique. A good correlation (P<0.001) was found with the plasma cell BrdU labelling index obtained with our reference immuno-enzymatic technique. Quantification of DNA content showed a satisfactory G1 peak coefficient of variation (CV) in diploid cells and a 4C to 2C ratio equal to 2. With this technique, the nuclear and cytoplasmic structures of both myeloid cells and plasma cells were well preserved, while their sensitivity to DNA denaturation was quite different.     

不同固定方法对植物细胞扫描电镜观察用冷冻断裂样品的影响

用冷冻断裂法在扫描电镜下研究了洋葱(Allium cepa)根端分生组织细胞内部的三维结构。采用了两种固定方法。冷冻断裂前只用1％锇酸固定的材料容易在细胞质和核之间断开,而用卡诺固定液(无水乙醇:冰醋酸3:1)前固定,然后再用1％锇酸固定的材料容易使细胞核断裂。前一固定方法适于研究细胞质的内部结构(细胞骨架的纤维、线粒体、内质网等及其三维分布关系):后一固定方法适于研究核内结构(染色质、核仁、核基质纤维)的三维形象,特别是核仁纤维中心染色质的三维结构。     

Effects of Different Fixatives on Freeze-Fractured Samples of Plant Cells for SEM Observations

Three-dimensional structures of meristematic cells of Allium cepa were studied using freeze-fracture method under the scanning electron microscope. Two fixation procedures were used. The cells were often fractured between eytoplasm and nucleus when the materials were fixed in 1% OsO4 alone before freeze fracture, whereas the nuclei, were frequently fractured if the materials were fixed first in Carnoy's, solution (ethanol: acetic acid=3:l) and then in 1% OsO4 before freeze fracture. The former fixation procedure is suitable for the study of the interior structures of cytoplasm such as cytoskeleton fibres, mitochondria, endoplasmic reticulum and their three-dimensional topography. The latter fixation method is suitable for the study of interior structures of nucleus such as chromatin, nucleoli, nuclear matrix filaments and their 3-dimensional architectures, especially the 3-dimensional structures of chromatin in fibrillar centre of the nucleolus.     

Fixation-dependent organization of core histones following DNA fluorescent in situ hybridization

We have evaluated the effects of different DNA denaturation protocols commonly used in DNA fluorescent in situ hybridization (FISH) experiments on chromatin structure using indirect immunofluorescence. The use of antibodies to acetylated histones H3 and H4 demonstrates that the different procedures differ considerably in their extent of histone displacement. Procedures involving paraformaldehyde fixation were found to be compatible with the structural preservation of acetylated chromatin organization by indirect immunofluorescence. These results provide a basis for interpreting DNA FISH experiments aimed at determining chromatin organization of individual loci. Received: 19 November 1996; in revised form: 10 January 1997 / Accepted: 10 January 1997     

A note on the demonstration of nuclear acidic HCl insoluble proteins in peripheral blood smears.

The present study deals with the conditions necessary for the specific demonstration of nuclear acidic HCl insoluble proteins is smeared peripheral leucocytes by means of a simple cytochemical procedures. These proteins can be very easily selectively demonstrated by staining with Azure C or Toluidine blue above pH 6, after the fixation of smears with methanol, extraction of histones, nucleic acids (with HCl, TCA) and treatment with formaldehyde. The double fixation with methanol and formaldehyde facilitated specific extraction of nuclear acidic HCl insoluble proteins of smeared leucocytes with pepsin. The nuclear acidic HCl insoluble proteins are present in leucocytes in interchromatin areas and their isoelectric point determined by cytochemical procedure is pH 6.     

The influence of fixation on the morphology of mitotic chromosomes

It is well known that there is a strong influence of fixation, i.e., acetic methanol versus formaldehyde, on the chromosome morphology at stages of the first meiotic division. In this study the influence of both these types of fixation on the morphology of mitotic chromosomes was examined in human lymphocytes. After methanol-acetic acid (3:1) fixation, the chromosomes show the "classical" condensed shape in which it is not always possible to recognize the two sister chromatids. These chromosomes are accessible to the conventional G-, R-, and C-banding techniques. After formaldehyde fixation at a relatively high pH, the chromosomes are thinner and longer (two to six times) when compared with chromosomes following methanol-acetic acid fixation. They show a scaffold-like morphology, sometimes with a halo of thin material around it. In all cases the two sister chromatids could be recognized. This chromosome structure could be easily stained with silver, Giemsa, 4,6-diamino-2-phenyl-indole (DAPI), and fluorescein isocyanate isomere 1 (FITC). The results obtained following these stainings gave no indication to any specific chemical composition of a probable central scaffold. The scaffold-like structures were not accessible to G-, R-, or C-banding techniques. The only effect observed following these banding techniques was the disappearance of the halo of thin material around the central scaffold-like structure.     

小鼠生精细胞染色质与染色体构筑的扫描电镜研究（简报）

It remains unclear about the intermediate construction of chromosome due to its highly compact nature and the limitation in methods. The present study was designed to investigate the construction of chromatin and mitotic chromosome in situ with scanning electron microscopy. Mouse testes were selected as the material, because of in which the spermatogenic cells divide actively and successively to form the sperm. Such a feature would be able to study the structure of mammalian chromatin and chromosomes along with the change of nuclear cycle. The animal were perfused with 200 ml of 0.075 mol/L KCl hypotonic solution to remove blood and placed for 15-20 min on ice followed by 0.5% glutaraldehyde and 0.5% formaldehyde for fixing. Through treated by the routine process of fractured and freeze dried with t-butyl alcohol, the specimens were then coated with a 3 nm thick platinum and observed with Hitachi S-430 scanning electron microscopy. It was found that the hypotonic treatment with 0.075 mol/L KCl solution was suit for demonstrating the nuclear structure, when the organelles were well preserved. The chromatin fibers of 10-30 nm and 80-125 nm in diameter could be recognized in the interphase nuclei, which were arranged losely at the region of euchromatin, and folded with each other into chromatin masses at the region of heterochromatin, while the chromatin fibers with the diameter of 80-125 nm often could be viewed on the mitotic chromosomes. Since its presence in interphase nuclei and mitotic chromosomes, it was considered that the chromatin fibers with 80-125 nm in diameter might play a role in the condensation of chromosome, serve as a type of the intermediate structure.     

Localization of proteins, specifically binding highly-repetitive sequences of DNA in human spermatozoids

Immunoblot revealed in spermatozoa alpha-satellite (sat) DNA-specific centromere protein B (CENP-B) and p70 (Enukashvily et al., 2000), a membrane telomere binding protein (MTBP/TRF2) (Podgornaya et al., 2000), and Alu-binding protein p68 (Lukyanov et al., 2000). The localization of some of these proteins in spermatozoa was defined using indirect immunofluorescence. Spermatozoa were fixed in methanol/acetic acid 3:1, or prior to fixation were treated with 5 mM heparin and 10 mM DTT. The heparin/DTT treatment causes the nuclear membrane destruction and a partial chromatin decondensation. In non-treated spermatozoa fluorescent signals from all ABs are registered near the membrane, with MTBP/TRF2 being localized closer to the acrosome than sat-DNA-specific proteins. In the treated spermatozoa MTBP/TRF2 was partially lost, whereas part of CENP-B and sat-p70 remained in contact with membrane. Another part of sat-binding proteins reveals a dot-like staining pattern, with dots confined to the DAPI-stained chromatin area, inside a nuclei. This is in partial agreement with the pattern of telomere and CEN position revealed by FISH. Commonly MTBP has a near membrane localization, being lost when the nuclear membrane is destroyed. Centromere-binding proteins are arranged in the order from the nuclear membrane towards the nuclear center, with CENP-B being situated more peripherally but not in the middle of the nucleus. This discrepancy may be explained by the fact, that some proteins are not associated with the appropriate sequences in a spermatozoon. Possibly, such a distribution of proteins may reflect their role in unpacking the paternal genetic material in a zygote.     

High-resolution detection of newly synthesized DNA by anti-bromodeoxyuridine antibodies identifies specific chromatin domains

We analyzed the incorporation of bromodeoxyuridine (BrdUrd) into DNA in exponentially growing murine erythroleukemia cells (FLC-745), using fluorescent anti-BrdUrd antibodies with light microscopy and flow cytometry. The fine localization of the DNA replicating sites was investigated at the ultrastructural level by using a second antibody conjugated with colloidal gold. The latter approach, which does not require acidic denaturation of the DNA, enables preservation of good morphology and obtains a better resolution power than that of electron microscopic autoradiography, the percentage of labeled cells obtained with the two techniques being comparable. After short BrdUrd pulses, characteristic distribution of the labeling can be identified in the heterochromatin, in interchromatin domains, or at the boundary between the dispersed and the condensed chromatin. Similar patterns are also observable in the nuclear structures which condense after acid denaturation, suggesting that DNA replication takes place at fixed sites associated with the nuclear matrix.     

Influence of cell fixatives and incubation schedule on PAP labeling of fibronectin in cultivated fibroblasts

A simple combined method for differential PAP labeling of fibronectin (FN) in mouse embryo fibroblast cultures was developed. Methanol-5% glacial acetic acid in dry ice-fixed cell monolayers showed mainly intracellular FN staining. Fixation with neutral paraformaldehyde before labeling, developed membrane- and extracellular matrix-associated FN. A combination of both procedures, which required incubation with primary antibody, fixation with paraformaldehyde followed by chilled acid methanol, and re-incubation with primary antibody, yielded sharp intracellular and extracellular FN labeling. The outlined methods can be readily employed in association with other staining techniques.     

Chromocenters of interphase nuclei in the mouse liver contain satellite DNA]

In isolated interphase mouse liver nuclei after hypotonic treatment only the chromocenters belonging to the pericentromeric heterochromatin remain in dense form while the main mass of a chromatin is completely decondensed. The centromeric nature of these chromocenters is demonstrated by their capability for C-banding and for hybridization with a satellite mouse DNA.     

Establishment of HEp-2 cell preparation for automated analysis of ANA fluorescence pattern.

BACKGROUND: Identification of antinuclear antibodies (ANAs) has large clinical importance for the assessment of autoimmune diseases. HEp-2 cell preparations on microscopic slides are commonly used as antigenic substrate. Methods used for cell preparation are important for ANA pattern analysis; however, these methods differ widely and are mostly not specified. METHODS: HEp-2 cells were fixed using acetic acid-ethanol, methanol-acetone, acetone, formaldehyde, paraformaldehyde, or glutaraldehyde. Morphological analysis was done after haematoxylin-eosin staining and DAPI-staining of cell nuclei. RESULTS: The results demonstrate a high variability of cell and nuclear morphology depending on the used fixatives. Aldehyde fixatives conserved the cell structures best, acetone fixatives revealed remarkable changes. CONCLUSIONS: After selecting appropriate fixation procedures to preserve nuclear structures further experiments are necessary to find out which fixation procedure preserves the disease-linked antigens the best way and are, therefore, suitable to be used in ANA-testing of AABs.     

PCR-based methylation testing for Prader-Willi or Angelman syndromes using archived fixed-cell suspensions

All Prader-Willi syndrome (PWS) and 75% of Angelman syndrome (AS) patients have specific DNA methylation pattern alterations that can be used for diagnostic evaluation. The methylation testing identifies a significantly higher proportion of patients as compared to fluorescence in situ hybridization (FISH)-based microdeletion analysis and is thus a useful diagnostic evaluation for clinically suspect, but FISH-negative, patients. We used two independent PCR-based protocols for methylation testing on fixed cell specimens archived after FISH analyses. Changes in DNA methylation due to the procedure of cell fixation were ruled out by testing control specimens before and after fixation. Then methylation testing was carried out on 20 standard fixed-cell supsensions from people suspected for PWS or AS. These fixed specimens were stored after negative FISH analysis for up to 4 years at 4 degrees C in 3:1 methanol/acetic acid. Methylation patterns associated with AS (one specimen) and with PWS (one specimen) were identified for both protocols. The observed methylation patterns were concordant with the phenotypes of the positive individuals and for the two protocols used. We have, thus, shown that archived fixed-cell suspensions from individuals suspected as PWS or AS that were negative for cytogenetic/FISH microdeletions, can now be re-evaluated with PCR-based methylation testing without the need for additional blood samples from the previously studied individuals.