Methanol-acetic Acid Fixation Research Articles

Active centromere and chromosome identification in fixed cell lines.

BackgroundThe centromere plays a crucial role in ensuring the fidelity of chromosome segregation during cell divisions. However, in cancer and constitutional disorders, the presence of more than one active centromere on a chromosome may be a contributing factor to chromosome instability and could also have predictive value in disease progression, making the detection of properly functioning centromeres important. Thus far, antibodies that are widely used for functional centromere detection mainly work on freshly harvested cells whereas most cytogenetic samples are stored long-term in methanol-acetic acid fixative. Hence, we aimed to identify antibodies that would recognise active centromere antigens on methanol-acetic acid fixed cells.ResultsA panel of active centromere protein antibodies was tested and we found that a rabbit monoclonal antibody against human CENP-C recognises the active centromeres of cells fixed in methanol-acetic acid. We then tested and compared combinations of established methods namely centromere fluorescence in situ hybridisation (cenFISH), centromere protein immunofluorescence (CENP-IF) and multicolour FISH (mFISH), and showed the usefulness of CENP-IF together with cenFISH followed by mFISH (CENP-IF-cenFISH-mFISH) with the aforementioned anti-CENP-C antibody. We further demonstrated the utility of our method in two cancer cell lines with high proportion of centromere defects namely neocentromere and functional dicentric.ConclusionsWe propose the incorporation of the CENP-IF-cenFISH-mFISH method using a commercially available rabbit monoclonal anti-CENP-C into established methods such as dicentric chromosome assay (DCA), prenatal karyotype screening in addition to constitutional and cancer karyotyping. This method will provide a more accurate assessment of centromere abnormality status in chromosome instability disorders.

Experimental characterization of methanol-acetic acid fixative sessile drop dynamics in dry and humid air by video imaging and interference analysis

Dynamics of methanol and acetic acid (3:1, v/v) fixative sessile drop is important for metaphase chromosomal spreads in cytogenetic assays. However, it has not been well characterized by biologists from a physical science point of view. In this work, a simple optical setup was built to record the fixative drop spreading and evaporation process. Drop film thickness, cross-sectional profile and surface thinning speed were constructed from the observed interference patterns to show evolution of the process in both dry and humid air. Surface thinning speed analysis at the drop center suggested different evaporation regimes. The ability of characterizing fluid behavior at a scale comparable to the size of cells by interference fringes is expected to facilitate further understanding of the metaphase spreading process at micro- and nano-scale.

Chromosome shattering: a mitotic catastrophe due to chromosome condensation failure

Chromosome shattering has been described as a special form of mitotic catastrophe, which occurs in cells with unrepaired DNA damage. The shattered chromosome phenotype was detected after application of a methanol/acetic acid (MAA) fixation protocol routinely used for the preparation of metaphase spreads. The corresponding phenotype in the living cell and the mechanism leading to this mitotic catastrophe have remained speculative so far. In the present study, we used V79 Chinese hamster cells, stably transfected with histone H2BmRFP for live-cell observations, and induced generalized chromosome shattering (GCS) by the synergistic effect of UV irradiation and caffeine posttreatment. We demonstrate that GCS can be derived from abnormal mitotic cells with a parachute-like chromatin configuration (PALCC) consisting of a bulky chromatin mass and extended chromatin fibers that tether centromeres at a remote, yet normally shaped spindle apparatus. This result hints at a chromosome condensation failure, yielding a "shattered" chromosome complement after MAA fixation. Live mitotic cells with PALCCs proceeded to interphase within a period similar to normal mitotic cells but did not divide. Instead they formed cells with highly abnormal nuclear configurations subject to apoptosis after several hours. We propose a factor depletion model where a limited pool of proteins is involved both in DNA repair and chromatin condensation. Chromosome condensation failure occurs when this pool becomes depleted.

Effect of colcemid exposure and methanol acetic acid fixation on human metaphase chromosome structure

Distribution of human lymphoid cells in the mitotic subphases, prophase, prometaphase, metaphase and anatelophase, was examined, when cells had been cultured and without colcemid exposure. A higher average length of chromosome no. I was found after overnight fixation than after short time fixation with 3:1 methanol acetic acid fixative. The probability of observing banded chromosomes on slides that had not been exposed to any postfixational band induction was higher after overnight fixation than after short time fixation. Since an increase in the extraction of histones caused by the methanol acetic acid fixation of metaphase cells led to an increase of average chromosome length as well as more frequent banding of chromosomes, it is suggested that not only non-histone proteins but also histones are involved in the formation of chromosome bands. In cells harvested without having been exposed to colcemid. most of the observed chromosomes were banded prometaphase chromosomes. It is suggested that the chromosome condensation process, which is delayed or partly inhibited when inhibitors of RNA or protein synthesis are added to the growth medium, continues as long as colcemid exposed cells remain arrested at the meta-anaphase border with free -SH groups available for oxidation.

Unfixed and fixed human chromosomes show different staining patterns after restriction endonuclease digestion

Restriction endonucleases (REs) have been widely used to produce banding patterns on chromosomes, but it remains uncertain to what extent the patterns are due to the sequence specificity of the enzymes, and to what extent chromatin structure influences the pattern of digestion. To throw light on this question, we have digested with restriction endonucleases unfixed chromosomes prepared in two different ways (isolated, and whole metaphase cells spread with a cytocentrifuge) and compared the results with those obtained on conventionally fixed chromosomes. Unfixed isolated chromosomes are easily destroyed by REs; after fixation with cold methanol, which produced minimal alteration to the chromatin structure, the chromosomes are resistant to the action of REs, and conventional methanol-acetic acid fixation is required to permit the induction of banding patterns by REs. Unfixed cytocentrifuge preparations, in which the chromosomes are still surrounded by cytoplasm, are much more resistant to the action of REs, and again banding patterns were only induced after methanol-acetic acid fixation. We conclude that the action of restriction endonucleases on chromosomes is strongly influenced by chromatin organisation, and that methanol-acetic acid fixation is required to permit the induction of conventional banding patterns on chromosomes.

Preservation of large-scale chromatin structure in FISH experiments

The nuclear organization of specific endogenous chromatin regions can be investigated only by fluorescence in situ hybridization (FISH). One of the two fixation procedures is typically applied: (1) buffered formaldehyde or (2) hypotonic shock with methanol acetic acid fixation followed by dropping of nuclei on glass slides and air drying. In this study, we compared the effects of these two procedures and some variations on nuclear morphology and on FISH signals. We analyzed mouse erythroleukemia and mouse embryonic stem cells because their clusters of subcentromeric heterochromatin provide an easy means to assess preservation of chromatin. Qualitative and quantitative analyses revealed that formaldehyde fixation provided good preservation of large-scale chromatin structures, while classical methanol acetic acid fixation after hypotonic treatment severely impaired nuclear shape and led to disruption of chromosome territories, heterochromatin structures, and large transgene arrays. Our data show that such preparations do not faithfully reflect in vivo nuclear architecture.Electronic supplementary materialSupplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00412-006-0084-2 and is accessible for authorized users.

Influence of Cell Fixation on Chromatin Topography

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.

Sister chromatid differentiation in 5-bromo-2?-deoxyuridine-substituted chromosomes: A study with DNA-specific ligands and monoclonal antibody to histone H2B

Human and Chinese hamster chromosomes were obtained from cells grown in the presence of 5-bromodeoxyuridine (BrdU) for (a) one replicative round, (b) two replicative rounds, (c) one replicative round followed by another round in thymidine and (d) the last period of synthetic phase. Untreated chromosomes and chromosomes treated with UV radiation after previous staining with 33258 Hoechst as photosensitizer were studied in order to investigate the mechanism(s) responsible for BrdU-induced sister chromatid differentiation (SCD). Metaphases were prepared by (1) standard methanol-acetic acid fixative for subsequent investigation with Giemsa or DNA-specific dyes such as ethidium bromide, acridine orange and monoclonal antibodies to double- or single-stranded DNA; (2) the procedure for observation under phase-contrast or electron microscopy; and (3) the cytospin method for subsequent immunoreaction with a monoclonal antibody to histone H2B. Our data exclude the possibility that the presence/absence of BrdU in the template strand might affect chromatin organization and thus resistance, while confirming that UV-induced DNA alteration is not sufficient, by itself, to explain SCD mechanism(s). That molecules other than DNA play a role in explaining SCD production is indicated by the fact that BrdU incorporation induces alterations in DNA-histone H2B interactions which, in turn, seem to produce structural variations in chromatin, possibly at the level of condensation, as monitored by phase-contrast and electron microscopy.

A simple method for improving the reproducibility of the R-banding technique.

Pretreatment of chromosomal preparations improves the reproducibility of R-banding. The pretreatment consists of covering slides with protein film made by a mixing of foetal calf serum and methanol acetic acid fixative. This procedure allows other banding techniques (GTG, Ag NOR).

Influence of Q- and G-banding on the Feulgen-stainability of human metaphase chromosomes.

In this paper, model experiments on chicken red blood cell nuclei are described concerning the influence of methanol-acetic acid fixation and irradiation at different wavelengths, with and without prior Atebrin staining on subsequent Feulgen-stainability. In addition, data are reported on the influence on Feulgen-stainability of Giemsa-banding procedures, illumination of unstained chromosomes at 220 and 515 nm and exposure of unstained and Atebrin-stained chromosomes to illumination at 440 nm. The ASG and especially the trypsin-Giemsa technique appeared to reduce markedly Feulgen-stainability. The same holds true for Atebrin fluorescence of chromosomes. The data are discussed in relation to their implications for the assumed cause of the Q- and G-banding phenomena. Techniques are described that allow reliable Feulgen DNA measurements of individual chromosomes after application of either G- or Q-banding.

GIEMSA STAINING OF HETEROCHROMATIC AREAS OF HUMAN CHROMOSOMES FOR IDENTIFICATION

A simple technique is described for differential staining of human chromosomes with Giemsa. The procedure involves DNA denaturation with a methanol-acetic acid fixative, and subsequent annealing using a saline solution. This technique has a number of advantages over quinacrine fluorescence, and gives a more distinct banding pattern. It is a rapid, inexpensive procedure and can be done with existing equipment and material in most cytogenetic laboratories.