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| PREPARATION OF CHROMOSOME SPREADS |
An ideal method of tissue preparation ensures both good specimen morphology and that the target molecules are in the optimum state for probe access and hybridization. DNA:DNA in situ hybridization is usually carried out on chromosome spread preparations where chromosome and nuclei are released from cells and spread on a glass microscope slide. This method yields well separated and enlarged chromosomes with good morphology which can be analyzed in transmitted light or fluorescence microscopes. As precipitating fixatives including alcohol:acetic acid are used, many proteins are destroyed and access of probes to the DNA within the spread chromosomes is very good. However, spreading looses cellular morphology and the three-dimensional organization of the nuclei and chromosomes. Spread preparations are used for mapping and analyzing the long-range organization of DNA sequences, and can be made from mitotic or meiotic material or DNA depending on the resolution required. Methods for spreading chromosomes are widely applicable to plants, animals and fungi and the following protocols can be adapted to suit all kinds of material. Methods for fixing, embedding and cutting sections, making whole mounts or centrifuge (Cytospin) preparations, used for most RNA in situ hybridization experiments and three dimensional DNA:DNA in situ hybridization studies are described in material.
High quality chromosome preparations are required for the best DNA:DNA in situ hybridization. In particular, remains of cytoplasm and other cellular and cell wall material will reduce the in situ hybridization signal and generate high levels of background. Ideally, cells and nuclei should be spread to a single layer and there should be little or no contact between cells and nuclei. Individual chromosomes from metaphase cells should be spread apart sufficiently so their number and morphology can be assessed easily. Nevertheless, useful results may come from material that is far from ideal; molecular cytogenetic methods have enabled for the first time cytogenetic analysis of material with no possibility of making metaphase preparations. Interphase nuclei from non-dividing tissues are often used to determine ploidy or aneuploidy with specific DNA probes and several hundred publications describe in situ hybridization to human cancers and amniotic fluids. Researchers have been able to analyze 30 year old biopsy material from wax sections, necrotic tissue cultures and biopsies, or highly differentiated cells using in situ hybridization.
Useful protocols about making chromosome preparations from different cells and for different purposes including karyotype analysis and chromosome banding are given by Macgregor & Varley (1988, for many animals), Schwarzacher & Wolf (1974, for human), Verma & Babu (1995, for human) and Fukui & Nakayama (1996, for plants).
The microscope slide The first requirement for chromosome preparation is the glass slide. Different batches and suppliers of glass clearly give different qualities of in situ hybridization results. With the acid treatment below, all slides seem to work successfully. Omitting this step often leads to selective loss of nuclei and metaphase spreads (see Protocol 5.8 and Fig. 5.1) and poor in situ hybridization results. The soaking modifies the surface properties of the glass so the spread material is not removed during subsequent treatments. Some slide makes (sometimes batches) may not need soaking
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